Polypeptides having peroxidase activity and nucleic acids encoding same

ABSTRACT

The present invention relates to isolated polypeptides having peroxidase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of pending U.S.application Ser. No. 09/596,824 filed Jun. 19, 2000, which applicationis fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to isolated polypeptides havingperoxidase activity and isolated nucleic acid sequences encoding thepolypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the nucleic acid sequences as well asmethods for producing and using the polypeptides.

[0004] 2. Description of the Related Art

[0005] Lignin is an aromatic polymer occurring in the woody tissue ofhigher plants. Due to its hydrophobicity and complex random structurelacking regular hydrolyzable bonds, lignin is poorly degraded by mostorganisms. The best degraders of lignin are white rot fungi that produceextracellular peroxidases and laccases, which are involved in theinitial attack of lignin.

[0006] Manganese-dependent peroxidase is a frequently encounteredperoxidase produced by white rot fungi. The peroxidase has a catalyticcycle involving a 2-electron oxidation of the heme by hydrogen peroxideand subsequent oxidation of compound I via compound II in two 1-electronsteps to the native enzyme. The best reducing substrate for compounds Iand II is Mn(II), a metal naturally present in wood. The Mn(III) formedoxidizes other substrates.

[0007] Organic acids such as oxalate, glyoxylate and lactate are knownto have an important role in the mechanism of manganese-dependentperoxidase and lignin degradation. Mn(III) is stripped from the enzymeby organic acids, and the produced Mn(III)-organic acid complex acts asa diffusible mediator in the oxidation of lignin by manganese-dependentperoxidase. Mn(III) can also oxidize organic acids, yielding radicals.The organic acids may also be supplied from the degradation of ligninand by microorganisms.

[0008] Field and Mester, 1998, Journal of Biochemistry 273: 15412-15417,disclose a manganese peroxidase-lignin peroxidase hybrid produced byBjerkandera sp. strain BOS55, which is able to oxidize varioussubstrates in the absence of manganese.

[0009] It is an object of the present invention to provide improvedpolypeptides having peroxidase activity and nucleic acid encoding thepolypeptides.

SUMMARY OF THE INVENTION

[0010] The present invention relates to isolated polypeptides havingperoxidase activity selected from the group consisting of:

[0011] (a) a polypeptide having an amino acid sequence which has atleast 75% identity with amino acids 22 to 370 of SEQ ID NO: 2, aminoacids 22 to 366 of SEQ ID NO: 4, or amino acids 19 to 362 of SEQ ID NO:6;

[0012] (b) a polypeptide encoded by a nucleic acid sequence whichhybridizes under medium-high stringency conditions with (i) nucleotides772 to 2302 of SEQ ID NO: 1, nucleotides 2008 to 3462 of SEQ ID NO: 3,or nucleotides 2848 to 4247 of SEQ ID NO: 5, (ii) the cDNA sequencecontained in nucleotides 772 to 2302 of SEQ ID NO: 1, nucleotides 2008to 3462 of SEQ ID NO: 3, or nucleotides 2848 to 4247 of SEQ ID NO: 5,(iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv)a complementary strand of (i), (ii), or (iii);

[0013] (c) a variant of the polypeptide having an amino acid sequence ofSEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, comprising a substitution,deletion, and/or insertion of one or more amino acids;

[0014] (d) an allelic variant of (a) or (b); and

[0015] (e) a fragment of (a), (b), or (d) that has peroxidase activity.

[0016] The present invention also relates to isolated nucleic acidsequences encoding the polypeptides and to nucleic acid constructs,vectors, and host cells comprising the nucleic acid sequences as well asmethods for producing and using the polypeptides.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIGS. 1A, B, and C show the genomic DNA sequence and the deducedamino acid sequence of a Bjerkandera adusta ATCC 90940 peroxidase (SEQID NOs: 1 and 2, respectively).

[0018]FIGS. 2A, B, C, and D show the genomic DNA sequence and thededuced amino acid sequence of a Bjerkandera adusta ATCC 90940peroxidase (SEQ ID NOs: 3 and 4, respectively).

[0019]FIGS. 3A, B, C, D, and E show the genomic DNA sequence and thededuced amino acid sequence of a Bjerkandera adusta ATCC 90940peroxidase (SEQ ID NOs: 5 and 6, respectively).

DETAILED DESCRIPTION OF THE INVENTION

[0020] Polypeptides Having Peroxidase Activity

[0021] The term “peroxidase activity” is defined herein as anoxidation-reduction activity that catalyzes the oxidation of a suitablereducing substrate (H⁺ donor) by hydrogen peroxide through the formationof a heme intermediate. When the reducing substrate is Mn(II) ion, theperoxidase activity is then specified as manganese peroxidase activity.

[0022] For purposes of the present invention, peroxidase activity ismeasured according to the procedure described by Mester and Field, 1998,Journal of Biological Chemistry 273: 15412-15417, where the oxidation ofMn(IT) is monitored by the formation of Mn(III)-malonate complex at 270nm or by the secondary oxidation of phenol red with Mn(II) at 600 nm andpH 4.5. Peroxidase activity may also be measured by monitoring theoxidation of 2,6-dimethoxyphenol to coerulignone, ABTS to ABTS+, andveratryl alcohol to veratraldehyde at 469, 420, and 310 nm,respectively, and pH 7.0. One unit of peroxidase activity is defined as1.0 μmole of hydrogen peroxidase consumed per minute at 25° C., pH 4.5or pH 7.

[0023] In a first embodiment, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofidentity to amino acids 22 to 370 of SEQ ID NO: 2, amino acids 22 to 366of SEQ ID NO: 4, or amino acids 19 to 362 of SEQ ID NO: 6 (i.e., themature polypeptide) of at least about 75%, preferably at least about80%, more preferably at least about 85%, even more preferably at leastabout 90%, most preferably at least about 95%, and even most preferablyat least about 97%, which have peroxidase activity (hereinafter“homologous polypeptides”). In a preferred embodiment, the homologouspolypeptides have an amino acid sequence which differs by five aminoacids, preferably by four amino acids, more preferably by three aminoacids, even more preferably by two amino acids, and most preferably byone amino acid from amino acids 22 to 370 of SEQ ID NO: 2, amino acids22 to 366 of SEQ ID NO: 4, or amino acids 19 to 362 of SEQ ID NO: 6. Forpurposes of the present invention, the degree of identity between twoamino acid sequences is determined by the Clustal method (Higgins, 1989,CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software (DNASTAR,Inc., Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters were Ktuple=1, gap penalty=3, windows=5,and diagonals=5.

[0024] Preferably, the polypeptides of the present invention comprisethe amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof;or a fragment thereof that has peroxidase activity. In a more preferredembodiment, the polypeptide of the present invention comprises the aminoacid sequence of SEQ ID NO: 2. In another preferred embodiment, thepolypeptide of the present invention comprises amino acids 22 to 370 ofSEQ ID NO: 2, or an allelic variant thereof; or a fragment thereof thathas peroxidase activity. In another preferred embodiment, thepolypeptide of the present invention comprises amino acids 22 to 370 ofSEQ ID NO: 2. In another preferred embodiment, the polypeptide of thepresent invention consists of the amino acid sequence of SEQ ID NO: 2 oran allelic variant thereof; or a fragment thereof that has peroxidaseactivity. In another preferred embodiment, the polypeptide of thepresent invention consists of the amino acid sequence of SEQ ID NO: 2.In another preferred embodiment, the polypeptide consists of amino acids22 to 370 of SEQ ID NO: 2 or an allelic variant thereof; or a fragmentthereof that has peroxidase activity. In another preferred embodiment,the polypeptide consists of amino acids 22 to 370 of SEQ ID NO: 2.

[0025] Preferably, the polypeptides of the present invention comprisethe amino acid sequence of SEQ ID NO: 4 or an allelic variant thereof;or a fragment thereof that has peroxidase activity. In a more preferredembodiment, the polypeptide of the present invention comprises the aminoacid sequence of SEQ ID NO: 4. In another preferred embodiment, thepolypeptide of the present invention comprises amino acids 22 to 366 ofSEQ ID NO: 4, or an allelic variant thereof; or a fragment thereof thathas peroxidase activity. In another preferred embodiment, thepolypeptide of the present invention comprises amino acids 22 to 366 ofSEQ ID NO: 4. In another preferred embodiment, the polypeptide of thepresent invention consists of the amino acid sequence of SEQ ID NO: 4 oran allelic variant thereof; or a fragment thereof that has peroxidaseactivity. In another preferred embodiment, the polypeptide of thepresent invention consists of the amino acid sequence of SEQ ID NO: 4.In another preferred embodiment, the polypeptide consists of amino acids22 to 366 of SEQ ID NO: 4 or an allelic variant thereof; or a fragmentthereof that has peroxidase activity. In another preferred embodiment,the polypeptide consists of amino acids 22 to 366 of SEQ ID NO: 4.

[0026] Preferably, the polypeptides of the present invention comprisethe amino acid sequence of SEQ ID NO: 6 or an allelic variant thereof;or a fragment thereof that has peroxidase activity. In a more preferredembodiment, the polypeptide of the present invention comprises the aminoacid sequence of SEQ ID NO: 6. in another preferred embodiment, thepolypeptide of the present invention comprises amino acids 19 to 362 ofSEQ ID NO: 6, or an allelic variant thereof; or a fragment thereof thathas peroxidase activity. In another preferred embodiment, thepolypeptide of the present invention comprises amino acids 19 to 362 ofSEQ ID NO: 6. In another preferred embodiment, the polypeptide of tiepresent invention consists of the amino acid sequence of SEQ ID NO: 6 oran allelic variant thereof; or a fragment thereof that has peroxidaseactivity. In another preferred embodiment, the polypeptide of thepresent invention consists of the amino acid sequence of SEQ ID NO: 6.In another preferred embodiment, the polypeptide consists of amino acids19 to 363 of SEQ ID NO: 6 or an allelic variant thereof; or a fragmentthereof that has peroxidase activity. In another preferred embodiment,the polypeptide consists of amino acids 19 to 363 of SEQ ID NO: 6.

[0027] A fragment of SEQ ID NO: 2 is a polypeptide having one or moreamino acids deleted from the amino and/or carboxyl terminus of thisamino acid sequence. Preferably, a fragment contains at least 295 aminoacid residues, more preferably at least 315 amino acid residues, andmost preferably at least 335 amino acid residues.

[0028] A fragment of SEQ ID NO: 4 is a polypeptide having one or moreamino acids deleted from the amino and/or carboxyl terminus of thisamino acid sequence. Preferably, a fragment contains at least 285 aminoacid residues, more preferably at least 305 amino acid residues, andmost preferably at least 325 amino acid residues.

[0029] A fragment of SEQ ID NO: 6 is a polypeptide having one or moreamino acids deleted from the amino and/or carboxyl terminus of thisamino acid sequence. Preferably, a fragment contains at least 285 aminoacid residues, more preferably at least 305 amino acid residues, andmost preferably at least 325 amino acid residues.

[0030] An allelic variant denotes any of two or more alternative formsof a gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

[0031] In a second embodiment, the present invention relates to isolatedpolypeptides having peroxidase activity which are encoded by nucleicacid sequences which hybridize under very low stringency conditions,preferably low stringency conditions, more preferably medium stringencyconditions, more preferably medium-high stringency conditions, even morepreferably high stringency conditions, and most preferably very highstringency conditions with a nucleic acid probe which hybridizes underthe same conditions with (i) nucleotides 772 to 2302 of SEQ ID NO: 1,nucleotides 2008 to 3462 of SEQ ID NO: 3, or nucleotides 2848 to 4247 ofSEQ ID NO: 5, (ii) the cDNA sequence contained in nucleotides 772 to2302 of SEQ ID NO: 1, nucleotides 2008 to 3462 of SEQ ID NO: 3, ornucleotides 2848 to 4247 of SEQ ID NO: 5, (iii) a subsequence of (i) or(ii), or (iv) a complementary strand of (i), (ii), or (iii) (J.Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). Thesubsequence of SEQ ID NO: 1 may be at least 100 nucleotides orpreferably at least 200 nucleotides. Moreover, the subsequence mayencode a polypeptide fragment which has peroxidase activity. Thepolypeptides may also be allelic variants or fragments of thepolypeptides that have peroxidase activity.

[0032] The nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQID NO: 5, or a subsequence thereof; as well as the amino acid sequenceof SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or a fragment thereof,may be used to design a nucleic acid probe to identify and clone DNAencoding polypeptides having peroxidase activity from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, preferably at least 25,and more preferably at least 35 nucleotides in length. Longer probes canalso be used. Both DNA and RNA probes can be used. The probes aretypically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

[0033] Thus, a genomic DNA or cDNA library prepared from such otherorganisms may be screened for DNA which hybridizes with the probesdescribed above and which encodes a polypeptide having peroxidaseactivity. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or asubsequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, the carriermaterial is used in a Southern blot. For purposes of the presentinvention, hybridization indicates that the nucleic acid sequencehybridizes to a labeled nucleic acid probe corresponding to the nucleicacid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, aswell as a complementary strand or a subsequence of these sequences,under very low to very high stringency conditions. Molecules to whichthe nucleic acid probe hybridizes under these conditions are detectedusing X-ray film.

[0034] In a preferred embodiment, the nucleic acid probe is a nucleicacid sequence which encodes the polypeptide of SEQ ID NO: 2, or asubsequence thereof. In another preferred embodiment, the nucleic acidprobe is SEQ ID NO: 1. In another preferred embodiment, the nucleic acidprobe is the mature polypeptide coding region of SEQ ID NO: 1. Inanother preferred embodiment, the nucleic acid probe is the nucleic acidsequence contained in plasmid pBM37-7 which is contained in Escherichiacoli NRRL B-30280, wherein the nucleic acid sequence encodes apolypeptide having peroxidase activity. In another preferred embodiment,the nucleic acid probe is the mature polypeptide coding region containedin plasmid pBM37-7 which is contained in Escherichia coli NRRL B-30280.

[0035] In a preferred embodiment, the nucleic acid probe is a nucleicacid sequence which encodes the polypeptide of SEQ ID NO: 4, or asubsequence thereof. In another preferred embodiment, the nucleic acidprobe is SEQ ID NO: 3. In another preferred embodiment, the nucleic acidprobe is the mature polypeptide coding region of SEQ ID NO: 3. Inanother preferred embodiment, the nucleic acid probe is the nucleic acidsequence contained in plasmid pBM38-1 which is contained in Escherichiacoli NRRL B-30281, wherein the nucleic acid sequence encodes apolypeptide having peroxidase activity. In another preferred embodiment,the nucleic acid probe is the mature polypeptide coding region containedin plasmid pBM38-1 which is contained in Escherichia coli NRRL B-30281.

[0036] In a preferred embodiment, the nucleic acid probe is a nucleicacid sequence which encodes the polypeptide of SEQ ID NO: 6, or asubsequence thereof. In another preferred embodiment, the nucleic acidprobe is SEQ ID NO: 5. In another preferred embodiment, the nucleic acidprobe is the mature polypeptide coding region of SEQ ID NO: 5. Inanother preferred embodiment, the nucleic acid probe is the nucleic acidsequence contained in plasmid pBM39-1 which is contained in Escherichiacoli NRRL, B-30282, wherein the nucleic acid sequence encodes apolypeptide having peroxidase activity. In another preferred embodiment,the nucleic acid probe is the mature polypeptide coding region containedin plasmid pBM39-1 which is contained in Escherichia coli NRRL B-30282.

[0037] For long probes of at least 100 nucleotides in length, very lowto very high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5× SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures.

[0038] For long probes of at least 100 nucleotides in length, thecarrier material is finally washed three times each for 15 minutes using2× SSC, 0.2% SDS preferably at least at 45° C. (very low stringency),more preferably at least at 50° C. (low stringency), more preferably atleast at 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

[0039] For short probes which are about 15 nucleotides to about 70nucleotides in length, stringency conditions are defined asprehybridization, hybridization, and washing post-hybridization at about5° C. to about 10° C. below the calculated T_(m) using the calculationaccording to Bolton and McCarthy (1962, Proceedings of the NationalAcademy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6,6 mM EDTA, 0.5% NP-40, 1× Denhardt's solution, 1 mM sodiumpyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mgof yeast RNA per ml following standard Southern blotting procedures.

[0040] For short probes which are about 15 nucleotides to about 70nucleotides in length, the carrier material is washed once in 6× SCCplus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6× SSCat 5° C. to 10° C. below the calculated T_(m).

[0041] In a third embodiment, the present invention relates to variantsof the polypeptide having an amino acid sequence of SEQ ID NO: 2, SEQ IDNO: 4, or SEQ ID NO: 6 comprising a substitution, deletion, and/orinsertion of one or more amino acids.

[0042] The amino acid sequences of the variant polypeptides may differfrom the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ IDNO: 6, or the mature polypeptides thereof, by an insertion or deletionof one or more amino acid residues and/or the substitution of one ormore amino acid residues by different amino acid residues. Preferably,amino acid changes are of a minor nature, that is conservative aminoacid substitutions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of one to about 30amino acids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

[0043] Examples of conservative substitutions are within the group ofbasic amino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these inreverse.

[0044] In a fourth embodiment, the present invention relates to isolatedpolypeptides having immunochemical identity or partial immunochemicalidentity to the polypeptide having the amino acid sequence of SEQ ID NO:2 or the mature polypeptide thereof. The immunochemical properties aredetermined by immunological cross-reaction identity tests by thewell-known Ouchterlony double immunodiffusion procedure. Specifically,an antiserum containing polyclonal antibodies which are immunoreactiveor bind to epitopes of the polypeptide having the amino acid sequence ofSEQ ID NO: 2 or the mature polypeptide thereof are prepared byimmunizing rabbits (or other rodents) according to the proceduredescribed by Harboe and Ingild, In N. H. Axelsen, J. Kroll, and B.Weeks, editors, A Manual of Quantitative Immunoelectrophoresis,Blackwell Scientific Publications, 1973, Chapter 23, or Johnstone andThorpe, Immunochemistry in Practice, Blackwell Scientific Publications,1982 (more specifically pages 27-31). A polypeptide havingimmunochemical identity is a polypeptide which reacts with the antiserumin an identical fashion such as total fusion of precipitates, identicalprecipitate morphology, and/or identical electrophoretic mobility usinga specific immunochemical technique. A further explanation ofimmunochemical identity is described by Axelsen, Bock, and Kroll, In N.H. Axelsen, J. Kroll, and B. Weeks, editors, A Manual of QuantitativeImmunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter10. A polypeptide having partial immunochemical identity is apolypeptide which reacts with the antiserum in a partially identicalfashion such as partial fusion of precipitates, partially identicalprecipitate morphology, and/or partially identical electrophoreticmobility using a specific immunochemical technique. A furtherexplanation of partial immunochemical identity is described by Bock andAxelsen, In N. H. Axelsen, J. Krøll, and B. Weeks, editors, A Manual ofQuantitative Immunoelectrophoresis, Blackwell Scientific Publications,1973, Chapter 11.

[0045] The antibody may also be a monoclonal antibody. Monoclonalantibodies may be prepared and used, e.g., according to the methods ofE. Harlow and D. Lane, editors, 1988, Antibodies, A Laboratory Manual,Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

[0046] The polypeptides of the present invention have at least 20%,preferably at least 40%, more preferably at least 60%, even morepreferably at least 80%, even more preferably at least 90%, and mostpreferably at least 100% of the peroxidase activity of the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.

[0047] A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by the nucleic acid sequence isproduced by the source or by a cell in which the nucleic acid sequencefrom the source has been inserted. In a preferred embodiment, thepolypeptide is secreted extracellularly.

[0048] A polypeptide of the present invention may be a fungalpolypeptide, and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide; or more preferably a filamentous fungal polypeptide such asan Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Cryptococcus,Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces,Polyporus, Schizophyllum, Talaromyces, Thermoascus, Thielavia,Tolypocladium, or Trichoderma polypeptide.

[0049] In a preferred embodiment, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis polypeptide.

[0050] In another preferred embodiment, the polypeptide is anAspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger,Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis, Fusariumcrookwellense, Fusarium culmorum, Fusarium graminearum, Fusariumgraminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusariumtorulosum, Fusarium trichothecioides, Fusarium venenatum, Humicolainsolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride polypeptide.

[0051] In another preferred embodiment, the polypeptide is a Bjerkanderaadusta, Bjerkandera fumosa, Polyporus adustus, Polyporus crispus,Polyporus halesiae, Polyporus fumosus, or Polyporus halesiaepolypeptide.

[0052] In a more preferred embodiment, the polypeptide is a Bjerkanderaadusta polypeptide, and most preferably a Bjerkandera adusta ATCC 90940polypeptide, e.g., the polypeptide with the amino acid sequence of SEQID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.

[0053] It will be understood that for the aforementioned species, theinvention encompasses both the perfect and imperfect states, and othertaxonomic equivalents, e.g., anamorphs, regardless of the species nameby which they are known. Those skilled in the art will readily recognizethe identity of appropriate equivalents. For example, taxonomicequivalents of Bjerkandera are defined by Farr et al., 1989, Fungi onPlants and Plant Products in the United States, APS Press, St. Paul,Minnesota. For instance, synonyms of Bjerkandera adusta are Polyporusadustus, Polyporus crispus, and Polyporus halesiae.

[0054] Strains of these species are readily accessible to the public ina number of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

[0055] Furthermore, such polypeptides may be identified and obtainedfrom other sources including microorganisms isolated from nature (e.g.,soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms from natural habitats are wellknown in the art. The nucleic acid sequence may then be derived bysimilarly screening a genomic or cDNA library of another microorganism.Once a nucleic acid sequence encoding a polypeptide has been detectedwith the probe(s), the sequence may be isolated or cloned by utilizingtechniques which are known to those of ordinary skill in the art(see,e.g., Sambrook et al., 1989, supra).

[0056] As defined herein, an “isolated” polypeptide is a polypeptidewhich is essentially free of other non-peroxidase polypeptides, e.g., atleast about 20% pure, preferably at least about 40% pure, morepreferably about 60% pure, even more preferably about 80% pure, mostpreferably about 90% pure, and even most preferably about 95% pure, asdetermined by SDS-PAGE.

[0057] Polypeptides encoded by nucleic acid sequences of the presentinvention also include fused polypeptides or cleavable fusionpolypeptides in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide or fragment thereof. A fusedpolypeptide is produced by fusing a nucleic acid sequence (or a portionthereof) encoding another polypeptide to a nucleic acid sequence (or aportion thereof) of the present invention. Techniques for producingfusion polypeptides are known in the art, and include ligating thecoding sequences encoding the polypeptides so that they are in frame andthat expression of the fused polypeptide is under control of the samepromoter(s) and terminator.

[0058] Nucleic Acid Sequences

[0059] The present invention also relates to isolated nucleic acidsequences which encode a polypeptide of the present invention. In apreferred embodiment, the nucleic acid sequence is set forth in SEQ IDNO: 1. In another more preferred embodiment, the nucleic acid sequenceis the sequence contained in plasmid pBM37-7 that is contained inEscherichia coli NRRL B-30280. In another preferred embodiment, thenucleic acid sequence is the mature polypeptide coding region of SEQ IDNO: 1. In another more preferred embodiment, the nucleic acid sequenceis the mature polypeptide coding region contained in plasmid pBM37-7that is contained in Escherichia coli NRRL B-30280. The presentinvention also encompasses nucleic acid sequences which encode apolypeptide having the amino acid sequence of SEQ ID NO: 2 or the maturepolypeptide thereof, which differ from SEQ ID NO: 1 by virtue of thedegeneracy of the genetic code. The present invention also relates tosubsequences of SEQ ID NO: 1 which encode fragments of SEQ ID NO: 2 thathave peroxidase activity.

[0060] In another preferred embodiment, the nucleic acid sequence is setforth in SEQ ID NO: 3. In another more preferred embodiment, the nucleicacid sequence is the sequence contained in plasmid pBM38-1 that iscontained in Escherichia coli NRRL B-30281. In another preferredembodiment, the nucleic acid sequence is the mature polypeptide codingregion of SEQ ID NO: 3. In another more preferred embodiment, thenucleic acid sequence is the mature polypeptide coding region containedin plasmid pBM38-1 that is contained in Escherichia coli NRRL B-30281.The present invention also encompasses nucleic acid sequences whichencode a polypeptide having the amino acid sequence of SEQ ID NO: 4 orthe mature polypeptide thereof, which differ from SEQ ID NO: 3 by virtueof the degeneracy of the genetic code. The present invention alsorelates to subsequences of SEQ ID NO: 3 which encode fragments of SEQ IDNO: 4 that have peroxidase activity.

[0061] In another preferred embodiment, the nucleic acid sequence is setforth in SEQ ID NO: 5. In another more preferred embodiment, the nucleicacid sequence is the sequence contained in plasmid pBM39-1 that iscontained in Escherichia coli NRRL B-30282. In another preferredembodiment, the nucleic acid sequence is the mature polypeptide codingregion of SEQ ID NO: 5. In another more preferred embodimert, thenucleic acid sequence is the mature polypeptide coding region containedin plasmid pBM39-1 that is contained in Escherichia coli NRRL B-30282.The present invention also encompasses nucleic acid sequences whichencode a polypeptide having the amino acid sequence of SEQ ID NO: 6 orthe mature polypeptide thereof, which differ from SEQ ID NO: 5 by virtueof the degeneracy of the genetic code. The present invention alsorelates to subsequences of SEQ ID NO: 5 which encode fragments of SEQ IDNO: 6 that have peroxidase activity.

[0062] A subsequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 is anucleic acid sequence encompassed by SEQ ID NO: 1, SEQ ID NO: 3, or SEQID NO: 5, respectively, except that one or more nucleotides from the 5′and/or 3′ end have been deleted. Preferably, a subsequence of SEQ ID NO:1 contains at least 885 nucleotides, more preferably at least 945nucleotides, and most preferably at least 1005 nucleotides. Preferably,a subsequence of SEQ ID NO: 3 contains at least 855 nucleotides, morepreferably at least 915 nucleotides, and most preferably at least 975nucleotides. Preferably, a subsequence of SEQ ID NO: 5 contains at least855 nucleotides, more preferably at least 915 nucleotides, and mostpreferably at least 975 nucleotides.

[0063] The present invention also relates to mutant nucleic acidsequences comprising at least one mutation in the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, in whichthe mutant nucleic acid sequence encodes a polypeptide which consists ofamino acids 22 to 370 of SEQ ID NO: 2, amino acids 22 to 366 of SEQ IDNO: 4, or amino acids 19 to 362 of SEQ ID NO: 6, respectively.

[0064] The techniques used to isolate or clone a nucleic acid sequenceencoding a polypeptide are known in the art and include isolation fromgenomic DNA, preparation from cDNA, or a combination thereof. Thecloning of the nucleic acid sequences of the present invention from suchgenomic DNA can be effected, e.g., by using the well known polymerasechain reaction (PCR) or antibody screening of expression libraries todetect cloned DNA fragments with shared structural features. See, e.g.,Innis et al., 1990, PCR: A Guide to Methods and Application, AcademicPress, New York. Other nucleic acid amplification procedures such asligase chain reaction (LCR), ligated activated transcription (LAT) andnucleic acid sequence-based amplification (NASBA) may be used. Thenucleic acid sequence may be cloned from a strain of Bjerkandera, oranother or related organism and thus, for example, may be an allelic orspecies variant of the polypeptide encoding region of the nucleic acidsequence.

[0065] The term “isolated nucleic acid sequence” as used herein refersto a nucleic acid sequence which is essentially free of other nucleicacid sequences, e.g., at least about 20% pure, preferably at least about40% pure, more preferably at least about 60% pure, even more preferablyat least about 80% pure, and most preferably at least about 90 % pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a hon cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

[0066] The present invention also relates to nucleic acid sequenceswhich have a degree of homology to the mature polypeptide codingsequence of SEQ ID NO: 1 (i.e., nucleotides 772 to 2302), SEQ ID NO: 3(i.e., nucleotides 2008 to 3462), SEQ ID NO: 5 (i.e, nucleotides 2848 to4247) of at least about 75%, preferably about 80%, preferably about 85%,more preferably about 90%, even more preferably about 95%, and mostpreferably about 97% homology, which encode an active polypeptide. Forpurposes of the present invention, the degree of homology between twonucleic acid sequences is determined by the Wilbur-Lipman method (Wilburand Lipman, 1983, Proceedings of the National Academy of Science USA 80:726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc.,Madison, WI) with an identity table and the following multiple alignmentparameters: Gap penalty of 10 and gap length penalty of 10. Pairwisealignment parameters were Ktuple=3, gap penalty=3, and windows=20.

[0067] Modification of a nucleic acid sequence encoding a polypeptide ofthe present invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., variantsthat differ in specific activity, thermostability, pH optimum, or thelike. The variant sequence may be constructed on the basis of thenucleic acid sequence presented as the polypeptide encoding part of SEQID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, e.g., a subsequence thereof,and/or by introduction of nucleotide substitutions which do not giverise to another amino acid sequence of the polypeptide encoded by thenucleic acid sequence, but which correspond to the codon usage of thehost organism intended for production of the enzyme, or by introductionof nucleotide substitutions which may give rise to a different aminoacid sequence. For a general description of nucleotide substitution,see, e.g. Ford et al., 1991, Protein Expression and Purification 2:95-107.

[0068] It will be apparent to those skilled in the art that suchsubstitutions can be made outside the regions critical to the functionof the molecule and still result in an active polypeptide. Amino acidresidues essential to the activity of the polypeptide encoded by theisolated nucleic acid sequence of the invention, and thereforepreferably not subject to substitution, may be identified according toprocedures known in the art, such as sitedirected mutagenesis oralanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, mutations areintroduced at every positively charged residue in the molecule, and theresultant mutant molecules are tested for peroxidase activity toidentify amino acid residues that are critical to the activity of themolecule. Sites of substrate-enzyme interaction can also be determinedby analysis of the three-dimensional structure as determined by suchtechniques as nuclear magnetic resonance analysis, crystallography orphotoaffinity labelling (see, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904;Wlodaver et al., 1992, FEBS Letters 309: 59-64).

[0069] The present invention also relates to isolated nucleic acidsequences encoding a polypeptide of the present invention, whichhybridize under very low stringency conditions, preferably lowstringency conditions, more preferably medium stringency conditions,more preferably medium-high stringency conditions, even more preferablyhigh stringency conditions, and most preferably very high stringencyconditions with a nucleic acid probe which hybridizes under the sameconditions with the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3,or SEQ ID NO: 5; or its complementary strand; or allelic variants andsubsequences thereof (Sambrook et al., 1989, supra), as defined herein.

[0070] The present invention also relates to isolated nucleic acidsequences produced by (a) hybridizing a DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i)nucleotides 772 to 2302 of SEQ ID NO: 1, nucleotides 2008 to 3462 of SEQID NO: 3, or nucleotides 2848 to 4247 of SEQ ID NO: 5, (ii) the cDNAsequence contained in nucleotides 772 to 2302 of SEQ ID NO: 1,nucleotides 2008 to 3462 of SEQ ID NO: 3, or nucleotides 2848 to 4247 ofSEQ ID NO: 5, (iii) a subsequence of (i) or (ii), or (iv) acomplementary strand of (i), (ii), or (iii); and (b) isolating thenucleic acid sequence. The subsequence is preferably a sequence of atleast 100 nucleotides such as a sequence which encodes a polypeptidefragment which has peroxidase activity.

[0071] Methods for Producing Mutant Nucleic Acid Sequences

[0072] The present invention further relates to methods for producing amutant nucleic acid sequence, comprising introducing at least onemutation into the mature polypeptide coding sequence of SEQ ID NO: 1,SEQ ID NO: 3, or SEQ ID NO: 5; or a subsequence thereof; wherein themutant nucleic acid sequence encodes a polypeptide which consists ofamino acids 22 to 370 of SEQ ID NO: 2, amino acids 22 to 366 of SEQ IDNO: 4, or amino acids 19 to 362 of SEQ ID NO: 6; or a fragment thereofwhich has peroxidase activity.

[0073] The introduction of a mutation into the nucleic acid sequence toexchange one nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure which utilizes a supercoiled,double stranded DNA vector with an insert of interest and two syntheticprimers containing the desired mutation. The oligonucleotide primers,each complementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with DpnI which isspecific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used.

[0074] Nucleic Acid Constructs

[0075] The present invention also relates to nucleic acid constructscomprising a nucleic acid sequence of the present invention operablylinked to one or more control sequences which direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences. Expression will be understood to include anystep involved in the production of the polypeptide including, but notlimited to, transcription, post-transcriptional modification,translation, post-translational modification, and secretion.

[0076] “Nucleic acid construct” is defined herein as a nucleic acidmolecule, either single- or double-stranded, which is isolated from anaturally occurring gene or which has been modified to contain segmentsof nucleic acid combined and juxtaposed in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term expression cassette when the nucleic acid constructcontains al′ the control sequences required for expression of a codingsequence of the present invention. The term “coding sequence” is definedherein as a nucleic acid sequence which directly specifies the aminoacid sequence of its protein product. The boundaries of a genomic codingsequence are generally determined by a ribosome binding site(prokaryotes) or by the ATG start codon (eukaryotes) located justupstream of the open reading frame at the 5′ end of the mRNA and atranscription terminator sequence located just downstream of the openreading frame at the 3′ end of the mRNA. A coding sequence can include,but is not limited to, DNA, cDNA, and recombinant nucleic acidsequences.

[0077] An isolated nucleic acid sequence encoding a polypeptide of thepresent invention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the nucleic acid sequenceprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifying nucleicacid sequences utilizing recombinant DNA methods are well known in theart.

[0078] The term “control sequences” is defined herein to include allcomponents which are necessary or advantageous for the expression of apolypeptide of the present invention. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleic acidsequence encoding a polypeptide. The term “operably linked” is definedherein as a configuration in which a control sequence is appropriatelyplaced at a position relative to the coding sequence of the DNA sequencesuch that the control sequence directs the expression of a polypeptide.

[0079] The control sequence may be an appropriate promoter sequence, anucleic acid sequence which is recognized by a host cell for expressionof the nucleic acid sequence. The promoter sequence containstranscriptional control sequences which mediate the expression of thepolypeptide. The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

[0080] Examples of suitable promoters for directing the transcription ofthe nucleic acid constructs of the present invention in a filamentousfungal host cell are promoters obtained from the genes for Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, and Fusarium oxysporum trypsin-like protease (WO 96/00787),as well as the NA2-tpi promoter (a hybrid of the promoters from thegenes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

[0081] In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL 1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8: 423-488.

[0082] The control sequence may also be a suitable transcriptionterminator sequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

[0083] Preferred terminators for filamentous fungal host cells areobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger glucoamylase, Aspergillus nidulans anthranilate synthase,Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-likeprotease.

[0084] Preferred terminators for yeast host cells are obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYCI), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

[0085] The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

[0086] Preferred leaders for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase and Aspergillusnidulans triose phosphate isomerase.

[0087] Suitable leaders for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

[0088] The control sequence may also be a polyadenylation sequence, asequence operably linked to the 3′ terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

[0089] Preferred polyadenylation sequences for filamentous fungal hostcells are obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

[0090] Useful polyadenylation sequences for yeast host cells aredescribed by Guo and Sherman, 1995, Molecular Cellular Biology 15:5983-5990.

[0091] The control sequence may also be a signal peptide coding regionthat codes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

[0092] Effective signal peptide coding regions for filamentous fungalhost cells are the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

[0093] In a preferred embodiment, the signal peptide coding region isnucleotides 709 to 771 of SEQ ID NO: 1 which encode amino acids 1 to 21of SEQ ID NO: 2; nucleotides 1893 to 2007 of SEQ ID NO: 3 which encodeamino acids 1 to 21 of SEQ ID NO: 4; or nucleotides 2794 to 2847 of SEQID NO: 5 which encode amino acids 1 to 18 of SEQ ID NO: 6.

[0094] Useful signal peptides for yeast host cells are obtained from thegenes for Saccharomyces cerevisiae alpha-factor and Saccharomycescerevisiae invertase. Other useful signal peptide coding regions aredescribed by Romanos et al, 1992, supra.

[0095] The control sequence may also be a propeptide coding region thatcodes for an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Saccharomyces cerevisiae alpha-factor, Rhizomucor mieheiaspartic proteinase, and Myceliophthora thermophila laccase (WO95/33836).

[0096] Where both signal peptide and propeptide regions are present atthe amino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

[0097] It may also be desirable to add regulatory sequences which allowhe regulation of the expression of the polypeptide relative to thegrowth of the host cell. Examples of regulatory systems are those whichcause the expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the TAKA alpha amylase promoter, Aspergillus nigerglucoamylase promoter, and Aspergillus oryzae glucoamylase promoter maybe used as regulatory sequences. Other examples of regulatory sequencesare those which allow for gene amplification. In eukaryotic systems,these include the dihydrofolate reductase gene which is amplified in thepresence of methotrexate, and the metallothionein genes which areamplified with heavy metals. In these cases, the nucleic acid sequenceencoding the polypeptide would be operably linked with the regulatorysequence.

[0098] The present invention also relates to nucleic acid constructs foraltering the expression of an endogenous gene encoding a polypeptide ofthe present invention. The constructs may contain the minimal number ofcomponents necessary for altering expression of the endogenous gene. Inone embodiment, the nucleic acid constructs preferably contain (a) atargeting sequence, (b) a regulatory sequence, (c) an exon, and (d) asplice-donor site. Upon introduction of the nucleic acid construct intoa cell, the construct inserts by homologous recombination into thecellular genome at the endogenous gene site. The targeting sequencedirects the integration of elements (a)-(d) into the endogenous genesuch that elements (b)-(d) are operably linked to the endogenous gene.In another embodiment, the nucleic acid constructs contain (a) atargeting sequence, (b) a regulatory sequence, (c) an exon, (d) asplice-donor site, (e) an intron, and (f) a splice-acceptor site,wherein the targeting sequence directs the integration of elements(a)-(f) such that elements (b)-(f) are operably linked to the endogenousgene. However, the constructs may contain additional components such asa selectable marker.

[0099] In both embodiments, the introduction of these components resultsin production of a new transcription unit in which expression of theendogenous gene is altered. In essence, the new transcription unit is afusion product of the sequences introduced by the targeting constructsand the endogenous gene. In one embodiment in which the endogenous geneis altered, the gene is activated. In this embodiment, homologousrecombination is used to replace, disrupt, or disable the regulatoryregion normally associated with the endogenous gene of a parent cellthrough the insertion of a regulatory sequence which causes the gene tobe expressed at higher levels than evident in the corresponding parentcell. The activated gene can be further amplified by the inclusion of anamplifiable selectable marker gene in the construct using methods wellknown in the art (see, for example, U.S. Pat. No. 5,641,670). In anotherembodiment in which the endogenous gene is altered, expression of thegene is reduced.

[0100] The targeting sequence can be within the endogenous gene,immediately adjacent to the gene, within an upstream gene, or upstreamof and at a distance from the endogenous gene. One or more targetingsequences can be used. For example, a circular plasmid or DNA fragmentpreferably employs a single targeting sequence, while a linear plasmidor DNA fragment preferably employs two targeting sequences.

[0101] The regulatory sequence of the construct can be comprised of oneor more promoters, enhancers, scaffold-attachment regions or matrixattachment sites, negative regulatory elements, transcription bindingsites, or combinations of these sequences.

[0102] The constructs further contain one or more exons of theendogenous gene. An exon is defined as a DNA sequence which is copiedinto RNA and is present in a mature mRNA molecule such that the exonsequence is in-frame with the coding region of the endogenous gene. Theexons can, optionally, contain DNA which encodes one or more amino acidsand/or partially encodes an amino acid. Alternatively, the exon containsDNA which corresponds to a 5′ non-encoding region. Where the exogenousexon or exons encode one or more amino acids and/or a portion of anamino acid, the nucleic acid construct is designed such that, upontranscription and splicing, the reading frame is in-frame with thecoding region of the endogenous gene so that the appropriate readingframe of the portion of the mRNA derived from the second exon isunchanged.

[0103] The splice-donor site of the constructs directs the splicing ofone exon to another exon. Typically, the first exon lies 5′ of thesecond exon, and the splice-donor site overlapping and flanking thefirst exon on its 3′ side recognizes a splice-acceptor site flanking thesecond exon on the 5′ side of the second exon. A splice-acceptor site,like a splice-donor site, is a sequence which directs the splicing ofone exon to another exon. Acting in conjunction with a splice-donorsite, the splicing apparatus uses a splice-acceptor site to effect theremoval of an intron.

[0104] Expression Vectors

[0105] The present invention also relates to recombinant expressionvectors comprising a nucleic acid sequence of the present invention, apromoter, and transcriptional and translational stop signals. Thevarious nucleic acid and control sequences described above may be joinedtogether to produce a recombinant expression vector which may includeone or more convenient restriction sites to allow for insertion orsubstitution of the nucleic acid sequence encoding the polypeptide atsuch sites. Alternatively, the nucleic acid sequence of the presentinvention may be expressed by inserting the nucleic acid sequence or anucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

[0106] The recombinant expression vector may be any vector (e.g., aplasmid or virus) which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the nucleic acidsequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

[0107] The vector may be an autonomously replicating vector, i.e., avector which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

[0108] The vectors of the present invention preferably contain one ormore selectable markers which permit easy selection of transformedcells. A selectable marker is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of suitable markers for yeast hostcells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectablemarkers for use in a filamentous fungal host cell include, but are notlimited to, amdS (acetamidase), argB (ornithine carbamoyltransferase),bar (phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-pliosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

[0109] The vectors of the present invention preferably contain anelement(s) that permits integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

[0110] For integration into the host cell genome, the vector may rely onthe nucleic acid sequence encoding the polypeptide or any other elementof the vector for integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 10,000 base pairs, preferably 400 to10,000 base pairs, and most preferably 800 to 10,000 base pairs, whichare highly homologous with the corresponding target sequence to enhancethe probability of homologous recombination. The integrational elementsmay be any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

[0111] For autonomous replication, the vector may further comprise anorigin of replication enabling the vector to replicate autonomously inthe host cell in question. Examples of origins of replication for use ina yeast host cell are the 2 micron origin of replication, ARS 1, ARS4,the combination of ARSI and CEN3, and the combination of ARS4 and CEN6.The origin of replication may be one having a mutation which makes itsfunctioning temperature sensitive in the host cell (see, e.g., Ehrlich,1978, Proceedings of the National Academy of Sciences USA 75: 1433).

[0112] More than one copy of a nucleic acid sequence of the presentinvention may be inserted into the host cell to increase production ofthe gene product. An increase in the copy number of the nucleic acidsequence can be obtained by integrating at least one additional copy ofthe sequence into the host cell genome or by including an amplifiableselectable marker gene with the nucleic acid sequence where cellscontaining amplified copies of the selectable marker gene, and therebyadditional copies of the nucleic acid sequence, can be selected for bycultivating the cells in the presence of the appropriate selectableagent.

[0113] The procedures used to ligate the elements described above toconstruct the recombinant expression vectors of the present inventionare well known to one skilled in the art (see, e.g., Sambrook et al.,1989, supra).

[0114] Host Cells

[0115] The present invention also relates to recombinant host cells,comprising a nucleic acid sequence of the invention, which areadvantageously used in the recombinant production of the polypeptides. Avector comprising a nucleic acid sequence of the present invention isintroduced into a host cell so that the vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication. The choice of a host cell will to a largeextent depend upon the gene encoding the polypeptide and its source.

[0116] The host cell may be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

[0117] In a preferred embodiment, the host cell is a fungal cell.“Fungi” as used herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

[0118] In a more preferred embodiment, the fungal host cell is a yeastcell. “Yeast” as used herein includes ascosporogenous yeast(Endomycetales), basidiosporogenous yeast, and yeast belonging to theFungi Imperfecti (Blastomycetes). Since the classification of yeast maychange in the future, for the purposes of this invention, yeast shall bedefined as described in Biology and Activities of Yeast (Skinner, F. A.,Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol.Symposium Series No. 9, 1980).

[0119] In an even more preferred embodiment, the yeast host cell is aCandida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia cell.

[0120] In a most preferred embodiment, the yeast host cell is aSaccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomycesdiastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,Saccharomyces norbensis or Saccharomyces oviformis cell. In another mostpreferred embodiment, the yeast host cell is a Kluyveromyces lactiscell. In another most preferred embodiment, the yeast host cell is aYarrowia lipolytica cell.

[0121] In another more preferred embodiment, the fungal host cell is afilamentous fungal cell. “Filamentous fungi” include all filamentousforms of the subdivision Eumycota and Oomycota (as defined by Hawksworthet al., 1995, supra). The filamentous fungi are generally characterizedby a mycelial wall composed of chitin, cellulose, glucan, chitosan,mannan, and other complex polysaccharides. Vegetative growth is byhyphal elongation and carbon catabolism is obligately aerobic. Incontrast, vegetative growth by yeasts such as Saccharomyces cerevisiaeis by budding of a unicellular thallus and carbon catabolism may befermentative.

[0122] In an even more preferred embodiment, the filamentous fungal hostcell is a cell of a species of, but not limited to, Acremonium,Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora,Penicillium, Thielavia, Tolypocladium, or Trichoderma.

[0123] In a most preferred embodiment, the filamentous fungal host cellis an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum cell. In an even mostpreferred embodiment, the filamentous fungal parent cell is a Fusariumvenenatum (Nirenberg sp. nov.) cell. In another most preferredembodiment, the filamentous fungal host cell is a Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0124] Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner knownper se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardieret al., 1989, Gene 78: 147-156, and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al, 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

[0125] Methods of Production

[0126] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating astrain, which in its wild-type form is capable of producing thepolypeptide, to produce the polypeptide; and (b) recovering thepolypeptide. Preferably, the strain is of the genus Bjerkandera, andmore preferably Bjerkandera adusta.

[0127] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

[0128] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleic acid sequence having atleast one mutation in the mature polypeptide coding region of SEQ ID NO:1, SEQ ID NO: 3, or SEQ ID NO: 5 wherein the mutant nucleic acidsequence encodes a polypeptide which consists of amino acids 22 to 370of SEQ ID NO: 2, amino acids 22 to 366 of SEQ ID NO: 4, or amino acids19 to 362 of SEQ ID NO: 6, respectively, and (b) recovering thepolypeptide.

[0129] The present invention further relates to methods for producing apolypeptide of the present invention comprising (a) cultivating ahomologously recombinant cell, having incorporated therein a newtranscription unit comprising a regulators sequence, an exon, and/or asplice donor site operably linked to a second exon of an endogenousnucleic acid sequence encoding the polypeptide, under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The methods are based on the use of gene activationtechnology, for example, as described in U.S. Pat. No. 5,641,670.

[0130] In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, and small-scale or large scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

[0131] The polypeptides may be detected using methods known in the artthat are specific for the polypeptides. These detection methods mayinclude use of specific antibodies, formation of an enzyme product, ordisappearance of an enzyme substrate. For example, an enzyme assay maybe used to determine the activity of the polypeptide as describedherein.

[0132] The resulting polypeptide may be recovered by methods known inthe art. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation,and/or precipitation.

[0133] The polypeptides of the present invention may be purified by avariety of procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J. -C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

[0134] Plants

[0135] The present invention also relates to a transgenic plant, plantpart, or plant cell which has been transformed with a nucleic acidsequence encoding a polypeptide having peroxidase activity of thepresent invention so as to express and produce the polypeptide inrecoverable quantities. The polypeptide may be recovered from the plantor plant parl. Alternatively, the plant or plant part containing therecombinant polypeptide may be used as such for improving the quality ofa food or feed, e.g., improving nutritional value, palatability, andrheological properties, or to destroy an antinutritive factor.

[0136] The transgenic plant can be dicotyledonous (a dicot) ormonocotyledonous (a monocot). Examples of monocot plants are grasses,such as meadow grass (blue grass, Poa), forage grass such as festuca,lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat,oats, rye, barley, rice, sorghum, and maize (corn).

[0137] Examples of dicot plants are tobacco, legumes, such as lupins,potato, sugar beet, pea, bean and soybean, and cruciferous plants(family Brassicaceae), such as cauliflower, rape seed, and the closelyrelated model organism Arabidopsis thaliana.

[0138] Examples of plant parts are stem, callus, leaves, root, fruits,seeds, and tubers. Also specific plant tissues, such as chloroplast,apoplast, mitochondria, vacuole, peroxisomes, and cytoplasm areconsidered to be a plant part. Furthermore, any plant cell, whatever thetissue origin, is considered to be a plant part.

[0139] Also included within the scope of the present invention are theprogeny of such plants, plant parts and plant cells.

[0140] The transgenic plant or plant cell expressing a polypeptide ofthe present invention may be constructed in accordance with methodsknown in the art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

[0141] Conveniently, the expression construct is a nucleic acidconstruct which comprises a nucleic acid sequence encoding a polypeptideof the present invention operably linked with appropriate regulatorysequences required for expression of the nucleic acid sequence in theplant or plant part of choice. Furthermore, the expression construct maycomprise a selectable marker useful for identifying host cells intowhich the expression construct has been integrated and DNA sequencesnecessary for introduction of the construct into the plant in question(the latter depends on the DNA introduction method to be used).

[0142] The choice of regulatory sequences, such as promoter andterminator sequences and optionally signal or transit sequences isdetermined, for example, on the basis of when, where, and how thepolypeptide is desired to be expressed. For instance, the expression ofthe gene encoding a polypeptide of the present invention may beconstitutive or inducible, or may be developmental, stage or tissuespecific, and the gene product may be targeted to a specific tissue orplant part such as seeds or leaves. Regulatory sequences are, forexample, described by Tague et al., 1988, Plant Physiology 86: 506.

[0143] For constitutive expression, the 35SCaMV promoter may be used(Franck et al., 1980, Cell 21: 285-294). Organ-specific promoters maybe, for example, a promoter from storage sink tissues such as seeds,potato tubers, and fruits (Edwards & Coruzzi, 1990,Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Itoet al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-71 1), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588).

[0144] A promoter enhancer element may also be used to achieve higherexpression of the enzyme in the plant. For instance, the promoterenhancer element may be an intron which is placed between the promoterand the nucleotide sequence encoding a polypeptide of the presentinvention. For instance, Xu et al., 1993, supra disclose the use of thefirst intron of the rice actin I gene to enhance expression.

[0145] The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

[0146] The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

[0147] Presently, Agrobacterium tumefaciens-mediated gene transfer isthe method of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38).However it can also be used for transforming monocots, although othertransformation methods are generally preferred for these plants.Presently, the method of choice for generating transgenic monocots isparticle bombardment (microscopic gold or tungsten particles coated withthe transforming DNA) of embryonic calli or developing embryos(Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994, CurrentOpinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

[0148] Following transformation, the transformants having incorporatedtherein the expression construct are selected and regenerated into wholeplants according to methods well-known in the art.

[0149] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleic acid sequenceencoding a polypeptide having peroxidase activity of the presentinvention under conditions conducive for production of the polypeptide;and (b) recovering the polypeptide.

[0150] Removal or Reduction of Peroxidase Activity

[0151] The present invention also relates to methods for producing amutant cell of a parent cell, which comprises disrupting or deleting anucleic acid sequence encoding the polypeptide or a control sequencethereof, which results in the mutant cell producing less of thepolypeptide than the parent cell when cultivated under the sameconditions.

[0152] The construction of strains which have reduced peroxidaseactivity may be conveniently accomplished by modification orinactivation of a nucleic acid sequence necessary for expression of thepolypeptide having peroxidase activity in the cell. The nucleic acidsequence to be modified or inactivated may be, for example, a nucleicacid sequence encoding the polypeptide or a part thereof essential forexhibiting peroxidase activity, or the nucleic acid sequence may have aregulatory function required for the expression of the polypeptide fromthe coding sequence of the nucleic acid sequence. An example of such aregulatory or control sequence may be a promoter sequence or afunctional part thereof, i.e., a part which is sufficient for affectingexpression of the polypeptide. Other control sequences for possiblemodification are described above.

[0153] Modification or inactivation of the nucleic acid sequence may beperformed by subjecting the cell to mutagenesis and selecting orscreening for cells in which the peroxidase producing capability hasbeen reduced. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

[0154] Examples of a physical or chemical mutagenizing agent suitablefor the present purpose include ultraviolet (UV) irradiation,hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodiumbisulphite, formic acid, and nucleotide analogues.

[0155] When such agents are used, the mutagenesis is typically performedby incubating the cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and selectingfor cells exhibiting reduced peroxidase activity or production.

[0156] Modification or inactivation of production of a polypeptide ofthe present invention may be accomplished by introduction, substitution,or removal of one or more nucleotides in the nucleic acid sequenceencoding the polypeptide or a regulatory element required for thetranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change of the open reading frame.Such modification or inactivation may be accomplished by site directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing the nucleicacid sequence to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

[0157] An example of a convenient way to eliminate or reduce productionby a host cell of choice is by gene replacement or gene interruption. Inthe gene interruption method, a nucleic acid sequence corresponding tothe endogenous gene or gene fragment of interest is mutagenized in vitroto produce a defective nucleic acid sequence which is then transformedinto the host cell to produce a defective gene. By homologousrecombination, the defective nucleic acid sequence replaces theendogenous gene or gene fragment. It may be desirable that the defectivegene or gene fragment also encodes a marker which may be used forselection of transformants in which the gene encoding the polypeptidehas been modified or destroyed.

[0158] Alternatively, modification or inactivation of the nucleic acidsequence may be performed by established anti-sense techniques using anucleotide sequence complementary to the polypeptide encoding sequence.More specifically, production of the polypeptide by a cell may bereduced or eliminated by introducing a nucleotide sequence complementaryto the nucleic acid sequence encoding the polypeptide which may betranscribed in the cell and is capable of hybridizing to the polypeptidemRNA produced in the cell. Under conditions allowing the complementaryanti-sense nucleotide sequence to hybridize to the polypeptide mRNA, theamount of polypeptide translated is thus reduced or eliminated.

[0159] It is preferred that the cell to be modified in accordance withthe methods of the present invention is of microbial origin, forexample, a fungal strain which is suitable for the production of desiredprotein products, either homologous or heterologous to the cell.

[0160] The present invention further relates to a mutant cell of aparent cell which comprises a disruption or deletion of a nucleic acidsequence encoding the polypeptide or a control sequence thereof, whichresults in the mutant cell producing less of the polypeptide than theparent cell.

[0161] The polypeptide-deficient mutant cells so created areparticularly useful as host cells for the expression of homologousand/or heterologous polypeptides. Therefore, the present inventionfurther relates to methods for producing a homologous or hetetologouspolypeptide comprising (a) cultivating the mutant cell under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The term “heterologous polypeptides” is defined herein aspolypeptides which are not native to the host cell, A native protein inwhich modifications have been made to alter the native sequence, or anative protein whose expression is quantitatively altered as a result ofa manipulation of the host cell by recombinant DNA techniques.

[0162] In a further aspect, the present invention relates to a methodfor producing a protein product essentially free of peroxidase activityby fermentation of a cell which produces both a polypeptide of thepresent invention as well as the protein product of interest by addingan effective amount of an agent capable of inhibiting peroxidaseactivity to the fermentation broth before, during, or after thefermentation has been completed, recovering the product of interest fromthe fermentation broth, and optionally subjecting the recovered productto further purification.

[0163] In a further aspect, the present invention relates to a methodfor producing a protein product essentially free of peroxidase activityby cultivating the cell under conditions permitting the expression ofthe product, subjecting the resultant culture broth to a combined pH andtemperature treatment so as to reduce the peroxidase activitysubstantially, and recovering the product from the culture broth.Alternatively, the combined pH and temperature treatment may beperformed on an enzyme preparation recovered from the culture broth. Thecombined pH and temperature treatment may optionally be used incombination with a treatment with a peroxidase inhibitor.

[0164] In accordance with this aspect of the invention, it is possibleto remove at least 60%, preferably at least 75%, more preferably atleast 85%, still more preferably at least 95%, and most preferably atleast 99% of the peroxidase activity. Complete removal of peroxidaseactivity may be obtained by use of this method.

[0165] The combined pH and temperature treatment is preferably carriedout at a pH in the range of 6.5-7 and a temperature in the range of25-40° C. for a sufficient period of time to attain the desired effect,where typically, 30 to 60 minutes is sufficient.

[0166] The methods used for cultivation and purification of the productof interest may be performed by methods known in the art.

[0167] The methods of the present invention for producing an essentiallyperoxidase-free product is of particular interest in the production ofeukaryotic polypeptides, in particular fungal proteins such as enzymes.The enzyme may be selected from, e.g., an amylolytic enzyme, lipolyticenzyme, proteolytic enzyme, cellulytic enzyme, oxidoreductase, or plantcell-wall degrading enzyme. Examples of such enzymes include anaminopeptidase, amylase, amyloglucosidase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, galactosidase,beta-galactosidase, glucoamylase, glucose oxidase, glucosidase,haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase,lipase, lyase, mannoidase, oxidase, pectinolytic enzyme, peroxidase,phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transferase, transglutaminase, or xylanase. Theperoxidase-deficient cells may also be used to express heterologousproteins of pharmaceutical interest such as hormones, growth factors,receptors, and the like.

[0168] It will be understood that the term “eukaryotic polypeptides”includes not only native polypeptides, but also those polypeptides,e.g., enzymes, which have been modified by amino acid substitutions,deletions or additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

[0169] In a further aspect, the present invention relates to a proteinproduct essentially free from peroxidase activity which is produced by amethod of the present invention.

[0170] Compositions

[0171] In a still further aspect, the present invention relates tocompositions comprising a polypeptide of the present invention and asuitable carrier. Preferably, the compositions are enriched in apolypeptide of the present invention. In the present context, the term“enriched” indicates that the peroxidase activity of the composition hasbeen increased, e.g., with an enrichment factor of 1.1.

[0172] The composition may comprise a polypeptide of the invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be producible by means of amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus niger, or Aspergillusoryzae, or Trichoderma, Humicola, preferably Humicola insolens, orFusarium, preferably Fusarium bactridioides, Fusarium cerealis, Fusariumcrookwellense, Fusarium culmorum, Fusarium graminearum, Fusariumgraminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusariumtrichothecioides, or Fusarium venenatum.

[0173] The polypeptide compositions may be prepared in accordance withmethods known in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

[0174] Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

[0175] Uses The present invention is also directed to methods for usingthe polypeptides having peroxidase activity.

[0176] The peroxidases can be used in number of different industrialprocesses. One process includes polymerization of lignin, both Kraft andlignosulfates, in order to produce a lignin with a higher molecularweight. A neutral/alkaline peroxidase is a particular advantage in thatKraft lignin is more soluble at higher pHs. Such methods are describedin, for example, Jin et al., 1991, Holzforschung 45: 467-468; U.S. Pat.No. 4,432,921; EP 0 275 544; PCT/DK93/00217, 1992. Peroxidase is alsouseful in the copolymerization of lignin with low molecular weightcompounds, such as is described by Milstein et al., 1994, Appl.Microbiol. Biotechnology 40: 760-767.

[0177] The peroxidases of the present invention can also be used forin-situ depolymerization of lignin in Kraft pulp, thereby producing apulp with lower lignin content. This use of peroxidase is an improvementover the current use of chlorine for depolymerization of lignin, whichleads to the production of chlorinated aromatic compounds, which are anenvironmentally undesirable by-product of paper mills. Such uses aredescribed in, for example, Current Opinion in Biotechnology 3: 261-266,1992; Journal of Biotechnology 25: 333-339, 1992; Hiroi et al., SvenskPapperstidning 5:162-166, 1976.

[0178] Oxidation of dyes or dye precursors and other chromophoriccompounds leads to decolorization of the compounds. Peroxidase can beused for this purpose, which can be particularly advantageous in asituation in which a dye transfer between fabrics is undesirable, e.g.,in the textile industry and in the detergent industry. Methods for dyetransfer inhibition and dye oxidation can be found in WO 92/01406; WO92/18683; WO 92/18687; WO 91/05839; EP 0495836; Calvo, 1991,Mededelingen van de Faculteit Landbouw-wetenschappen/RijiksuniversitetGent. 56: 1565-1567; Tsujino et al., 1991, J. Soc. Chem. 42: 273-282.Use of peroxidase in oxidation of dye precursors for hair dyeing isdisclosed in U.S. Pat. No. 3,251,742, the contents of which areincorporated herein by reference.

[0179] The present peroxidases can also be used for the polymerizationor oxidation of phenolic compounds present in liquids. An example ofsuch utility is the treatment of juices, such as apple juice, so thatthe peroxidase will accelerate a precipitation of the phenolic compoundspresent in the juice, thereby producing a more stable juice. Suchapplications have been described in Stutz, 1993, Fruit Processing 7/93,248-252; Maier et al., 1990, Dt. Lebensmittel-rindschau 86: 137-142;Dietrich et al., 1990, Fluss. Obst 57: 67-73.

[0180] Peroxidases of the present invention are also useful in soildetoxification (Nannipieri et al., 1991, J. Environ. Qual. 20: 510-517;Dec and Bollag, 1990, Arch. Environ. Contam. Toxicol. 19: 543-550).

[0181] Signal Peptide

[0182] The present invention also relates to nucleic acid constructscomprising a gene encoding a protein operably linked to a nucleic acidsequence consisting of nucleotides 709 to 771 of SEQ ID NO: 1,nucleotides 1893 to 2007 of SEQ ID NO: 3, or nucleotides 2794 to 2847 ofSEQ ID NO: 5 encoding a signal peptide consisting of amino acids 1 to 21of SEQ ID NO: 2, amino acids 1 to 21 of SEQ ID NO: 4, or amino acids 1to 18 of SEQ ID NO: 6, respectively, wherein the gene is foreign to thenucleic acid sequence.

[0183] The present invention also relates to recombinant expressionvectors and recombinant host cells comprising such nucleic acidconstructs.

[0184] The present invention also relates to methods for producing aprotein comprising (a) cultivating such a recombinant host cell underconditions suitable for production of the protein; and (b) recoveringthe protein.

[0185] The nucleic acid sequence may be operably linked to foreign geneswith other control sequences. Such other control sequences are describedsupra. As noted earlier, where both signal peptide and propeptideregions are present at the amino terminus of a protein, the propeptideregion is positioned next to the amino terminus of a protein and thesignal peptide region is positioned next to the amino terminus of thepropeptide region.

[0186] The protein may be native or heterologous to a host cell. Theterm “protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides which comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more may be heterologous or native to the host cell.Proteins further include naturally occurring allelic and engineeredvariations of the above mentioned proteins and hybrid proteins.

[0187] Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred embodiment, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred embodiment, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

[0188] The gene may be obtained from any prokaryotic, eukaryotic, orother source.

[0189] The present invention is further described by the followingexamples which should not be construed as limiting the scope of theinvention.

EXAMPLES

[0190] Chemicals used as buffers and substrates were commercial productsof at least reagent grade.

[0191] Fungal Strain

[0192]Bjerkandera adusta strain ATCC 90940 was used as the source ofgenomic DNA.

[0193] Bacterial Strains and Cloning Vectors

[0194] Plasmid pCR2.1-TOPO (Invitrogen, San Diego, Calif.) was used forthe cloning of the PCR amplified peroxidase gene fragments. Escherichiacoli TOPI0 (Invitrogen, San Diego, Calif.) was used as the host forcloning and maintenance of PCR products.

[0195] Plasmid pBluescript II KS(-) (Stratagene, La Jolla, Calif.) wasused for the cloning of the partial HindIII digested genomic DNA fromBjerkandera adusta ATCC 90940. E. coli SoloPack Gold supercompetentcells (Stratagene, LaJolla, Calif.) and XL10-Gold Kan ultracompetentcells (Stratagene, LaJolla, Calif.) were used to construct the partialBjerkandera adusta genomic library.

[0196] Media

[0197]Bjerkandera adusta strain ATCC 90940 was grown in YEG mediacomposed of 1% yeast extract and 2% peptone.

Example 1 Genomic DNA Isolation

[0198] Genomic DNA was prepared from Bjerkandera adusta strain ATCC90940 using a Qiagen Maxi Column (Qiagen, Valencia, Calif.) according tothe manufactuer's protocol. The mycelia were collected through Miracloth(Calbiochem, La Jolla, Calif.) and rinsed twice with TE buffer (10 mMTris-1 mM EDTA). The mycelia were frozen in liquid nitrogen and groundto a fine powder in an electric coffee grinder that was prechilled withdry ice. Approximately two grams of powder were transferred to adisposable 50 ml conical tube followed by 20 ml of lysis buffer (100 mMEDTA, 10 mM Tris, 1% Triton X-100, 500 mM guanidine-HCl, 200 mM NaCl pH5.0). The mixture was incubated at 37° C. for 30 minutes after additionof 20 μg of DNase-free RNase per ml. Proteinase K was added at 0.8 mg/mland incubated at 50° C. for 2 hours. A Qiagen Maxi column (Qiagen,Valencia, Calif.) was equilibrated with 10 ml of QBT buffer (Qiagen,Valencia, Calif.). The cleared lysate was transferred to the column tobind genomic DNA. The column was washed twice with 30 ml of QC buffer.DNA was eluted with 15 ml of QF buffer (Qiagen, Valencia, Calif.). A10.5 ml volume of room temperature filter-sterilized isopropanol wasadded and mixed gently. The DNA was pelleted by centrifugation at 1500×g for 20 minutes. The pellet was washed with ice-cold 70% ethanol andthen air-dried. The dried pellet was resuspended in TE buffer.

Example 2 PCR Amplification

[0199] Degenerate primers were designed based on the reported N-terminalamino acid sequence of a reported hybrid manganese/lignin peroxidasehybrid enzyme (Field and Mester, 1998, supra) and to conserved regions(“NCPGAPQ” and “RTACEWQ”) found in fungal lignin and manganeseperoxidases from various wood-degrading fungi. These primers were usedto PCR amplify a peroxidase from Bjerkandera adusta.

[0200] Set1: Primer 990355: 5′-GTIGCITGYCCIGAYGGIGTIAAYAC-3′ (SEQ ID NO:7) Primer 990356: 5′-TGIGGIGCICCIGGRCARTT-3′ (SEQ ID NO: 8)

[0201] Set2: Primer 990355: 5′-GTIGCITGYCCIGAYGGIGTIAAYAC-3′ (SEQ ID NO:9) Primer 990357: 5′-TGCCAYTCRCAIGCIGTIC-3′ (SEQ ID NO: 10)

[0202] PCR reactions (50 μl) were composed of 1.16 μg of Bjerkanderaadusta genomic DNA as the template, 50 pmoles of primer set 1 or 2, and1× HotStarTaq DNA polymerase Master Mix (Qiagen, Valencia, Calif.). Thereactions were performed in an Ericomp Twin Block System Easy Cyclerprogrammed as follows: Cycle 1 at 95° C. for 15 minutes; cycles 2-31each at 95° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 2minutes; and cycle 32 at 72° C. for 10 minutes.

[0203] A 10 μl volume of each PCR reaction was electrophoresed for 1hour at 100 volts on a 1% agarose gel using TAE buffer (40 mM Tris base,20 mM sodium acetate, 1 mM disodium EDTA pH 7.2). The results revealedthe presence of a PCR product of ˜540 bp from primer set 1 and a PCRproduct of ˜1050 bp from primer set 2.

[0204] The PCR product from primer set 1 (˜540 bp) and the PCR product(˜1050 bp) from primer set 2 were cloned into plasmid pCR2.1-TOPO andtransformed into Invitrogen TOP10 cells according to the manufacturer'sinstructions.

[0205] Analysis of the TOPO clones was performed by DNA sequencing usinga Perkin-Elmer Applied Biosystems Model 377 Sequencer XL (PerkinElmer/Applied Biosystems Inc., Foster City, Calif.) with dye-terminatorchemistry (Giesecke et al., 1992, Journal of Virology Methods 38: 47-60)and M13-forward and M13-reverse sequencing primers.

[0206] Sequence analysis suggested three independent clones, which weredesignated pBM37 (˜540 bp insert), pBM38 (˜540 bp insert), and pBM39(˜1050 bp insert).

Example 3 Southern Blot Analysis

[0207] Genomic DNA was analyzed by Southern Hybridization (Maniatis etal., 1982, Molecular Cloning, a Laboratory Manual, Cold Spring HarborPress, Cold Spring Harbor, N.Y.). Approximately 1 μg of Bjerkanderaadusta genomic DNA, prepared as described in Example 1, was digestedseparately with EcoRI, BamHI, HindIII, and SalI (Boehringer Mannheim,Indianapolis, Ind.) and fractionated by size on a 0.7% agarose gel usingTAE buffer. The gel was photographed under short wavelength UV light andsoaked twice for 15 minutes in 0.2 N HCl, followed by a brief rinse indouble distilled water. DNA was transferred to a Hybond N+ hybridizationmembrane (Amersham Life Science, Arlington Heights, IL) by capillaryblotting with 0.4 M NaOH using the Turbo Blot Method (Schleicher andSchuell, Keene, N.H.). The membrane was UV crosslinked (UV Stratalinker2400, Stratagene, La Jolla, Calif.) and incubated for 2 hours in thefollowing hybridization buffer at 65° C. with gentle agitation: 6× SSPE,7% SDS.

[0208] pBM37, pBM38, and pBM39 were digested with EcoRI andelectrophoresed on a 1% agarose gel using TAE buffer. Gene specificfragments from pBM37 (˜540 bp), pBM38 (˜540 bp), and pBM39 (˜1040 bp)were gel purified using a Qiagen Gel Extraction Kit (Qiagen, Valencia,Calif.) followed by radiolabeling using random priming (Prime it II,Stratagene, La Jolla, Calif.). The radiolabeled fragment probes wereadded to the hybridization buffer at an activity of approximately 1×10⁶cpm per ml of buffer. The mixture was incubated with the membraneovernight at 65° C. in a shaking water bath. Following incubation, themembrane was washed twice for 15 minutes in 2× SSC with 0.1% SDS at 65°C., and once in 2× SSC for 15 minutes at room temperature. The membranewas wrapped in plastic wrap and exposed to X-ray film for 24 hours at˜-80° C. with intensifying screens (Kodak, Rochester, N.Y.).

[0209] All three fragments hybridized to DNA digested with HindIII. ThepBM37 fragment hybridized to a 8.0 kb band, the pBM39 fragment to a 4.7kb band, and the pBM38 fragment to a3.9kb band.

Example 4 Bjerkandera adusta Partial Genomic Library Construction andScreening of the Library for Peroxidase Genomic Clones

[0210] A partial genomic library was constructed by partially digestingBjerkandera adusta ATCC 90940 genomic DNA with HindIII. Thirty units ofHindIII were used to digest 5.2 μg of Bjerkandera adusta genomic DNAusing conditions recommended by the manufacturer. The reaction wascarried out at 37° C., and samples were taken at 3 minute intervals from17 to 30 minutes. The reactions were placed on ice and stopped byaddition of 10× DNA loading dye (25% glycerol, 10 mM Tris pH 7.0, 10 mMEDTA, 0.025% bromophenol blue, 0.025% xylene cyanol). These digests weresize fractionated on a 0.7% agarose gel using TAE buffer, and the regionof the gel containing DNA ranging in size from 3 to 9 kb was excised.The excised DNA was gel purified using a Qiagen Gel Extraction kit(Qiagen, Valencia, Calif.) according to manufacturer's suggestions.

[0211] The size selected DNA was ligated into pBluescript II KS(−)previously digested with HindIII, and transformed into Solopack GoldSupercompetent cells and XL10-Gold Kan Ultracompetent cells.

[0212] The genomic library was screened to obtain genomic clones ofperoxidase genes. The library was plated to obtain approximately 1000colonies per 150-mm petri plate. The colonies were lifted to Hybond-N⁺hybridization filters (Amersham Life Science, Arlington Heights, Ill.)using standard protocols (Samsbrook et al., 1989, supra). The filterswere crosslinked with UV (UV Stratalinker 2400, Stratagene, La Jolla,Calif.) and incubated for 2 hours in 6× SSPE, 7% SDS at 65° C. withgentle agitation. The radiolabeled probes from pBM37, pBM38, and pBM39,as described in Example 3, were addec to the hybridization buffer at anactivity of approximately 1×10⁶ cpm per ml of buffer. The mixture wasincubated with the filters overnight at 65° C. in a shaking water bath.Following incubation, the filters were washed twice for fifteen minuteseach time in 2× SSC with 0.1% SDS at 65° C., and once in 2× SSC forfifteen minutes at room temperature. The filters were wrapped in plasticwrap and exposed to X-ray film for 24 hours at ˜80° C. with intensifyingscreens.

[0213] Several positive colonies were identified and purified tohomogeneity using standard protocols (Sambrook et al.,1989, supra). Thethree new clones were designated pBM37-7, pBM38-1 and pBM39-1,respectively.

Example 5 Analysis of Genomic Clones

[0214] DNA sequencing was performed with an Applied Biosystems Model 377Sequencer XL using dye-terminator chemistry. Complete nucleotidesequences were generated using a transposon insertion strategy using aPrimer Island Transposition Kit (Perkin Elmer/Applied Biosystems Inc.,Foster City, Calif.).

[0215] The first clone (pBM37-7) was sequenced to a redundancy of 6.4.The nucleotide sequence and deduced amino acid sequence are shown inFIG. 1. The first 25 amino acids were identical to the N-terminal aminoacid sequence of a hybrid manganese/lignin peroxidase from Bjerkanderaadusta (Field and Mester, 1998, supra). The insert contained an openreading frame of 1110 bp encoding a protein of 370 amino acids. Usingthe SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6),a signal peptide of 21 amino acid residues was predicted indicating themature peroxidase contains 349 amino acids. There is one potentialN-linked glycosylation site. Within the promoter region of clonepBM37-7, putative TATAAA and CAAT motifs can be found at positions −72and-353, respectively.

[0216] The positions of introns and exons within the pBM37-7 peroxidasegene were assigned based on alignments of the deduced amino acidsequence to the other filamentous fungal peroxidase gene products. Onthe basis of this comparison, the Bjerkandera adusta ATCC 90940 pBM37-7peroxidase gene is comprised of 10 exons (77, 138, 57, 135, 68, 54, 79,126, 310, and 69 bp) which are interrupted by 9 small introns (49, 52,53, 59, 56, 54, 53, 54, and 54 bp).

[0217] The pBM37-7 peroxidase gene has been deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, as E. coli NRRL B-30280.

[0218] The second clone (pBM38-1) was sequenced to a redundancy of 6.2.The nucleotide sequence and deduced amino acid sequence are shown inFIG. 2. The insert contained an open reading frame of 1098 bp encoding aprotein of 366 amino acids. Using the SignalP program, a signal peptideof 21 residues was predicted indicating the mature peroxidase contains345 amino acids. There are two potential N-linked glycosylation sites(Asn-X-Ser/Thr). Within the promoter region of clone pBM38-1, putativeTATAAA and CAAT motifs can be found at positions -73 and -112,respectively.

[0219] The positions of introns and exons within the pBM38-1 peroxidasegene were assigned based on alignments of the deduced amino acidsequence to other filamentous fungal peroxidase gene products. On thebasis of this comparison, the Bjerkandera adusta ATCC 90940 pBM38-1peroxidase gene is comprised of 10 exons (60, 151, 57, 135, 68, 133,124, 124, 176, and 69 bp) which are interrupted by 9 small introns (53,54, 53, 50, 51, 55, 47, 55, and 58 bp).

[0220] The pBM38-1 peroxidase gene has been deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, as E. coli NRRL B-30281.

[0221] The third clone (pBM39-1) was sequenced to a redundancy of 6.0.The nucleotide sequence and deduced amino acid sequence are shown inFIG. 3. The insert contained an open reading frame of 1086 bp encoding aprotein of 362 amino acids. Using the SignalP program, a signal peptideof 18 residues was predicted indicating the mature predicted peroxidasecontains 344 amino acids. There are three potential N-linkedglycosylation sites (Asn-X-Ser/Thr). The promoter region for this genecontains a TATAAA motif at position −89, but does not contain a CAATmotif.

[0222] The positions of introns and exons within the pBM39-1 peroxidasegene were assigned based on alignments of the deduced amino acidsequence to other filamentous fungal peroxidase gene products. On thebasis of this comparison, the Bjerkandera adusta ATCC 90940 peroxidasegene is comprised of 8 exons (61, 151, 24, 172, 68, 130, 242, and 245bp) which are interrupted by 7 small introns (52, 50, 45, 50, 53, 56,and 58 bp).

[0223] The pBM39-1 peroxidase gene has been deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, as E. coli NRRL B-30282.

[0224] A comparative alignment showed that deduced amino acid sequencesof the pBM37-7 peroxidase gene shares 63.6% identity with a ligninperoxidase from Phanerochaete chrysosporium (Swissprot P11542).

[0225] A comparative alignment showed that the deduced amino acidsequences of the pBM38-1 and pBM39-1 peroxidases share 62.4% and 55.7%identity, respectively, with a lignin peroxidase from Trametesversicolor (Swissprot P20013).

Deposit of Biological Material

[0226] The following biological material has been deposited under theterms of the Budapest Treaty with the Agricultural Research ServicePatent Culture Collection, Northern Regional Research Center, 1815University Street, Peoria, Ill., 61604, and given the followingaccession number: Deposit Accession Number Date of Deposit E. colipBM37-7 NRRL B-30280 April 24, 2000 E. coli pBM38-1 NRRL B-30281 April24, 2000 E. coli pBM39-1 NRRL B-30282 April 24, 2000

[0227] The strain has been deposited under conditions that assure thataccess to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

[0228] The invention described and claimed herein is not to be limitedin scope by the specific embodiments herein disclosed, since theseembodiments are intended as illustrations of several aspects of theinvention. Any equivalent embodiments are intended to be within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims. In the case of conflict, the present disclosure includingdefinitions will control.

[0229] Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1 10 1 2489 DNA Bjerkandera adusta misc_feature (1)...(2489) k = G or T1 ccatggtatg tcgtttggtt ccgtcgggac agttcgagtt cgccgagaac ggtgcgtccc 60gcctttgaat actctgaatg gccccgttaa tgaatgtcca ttcagatgaa gcgcgagagg 120tggtaccttg tagagctcac atccaactgt cccgacgcac tggaattgtc acaaggccct 180agactttggg tccctaggac gtcaaaaaac cgtgttcgac ctgcgtctta caaccttccg 240atgtttctgt tgacaccggc aggaacgata acgtgtgtag agctccaagt ttagttggcc 300caacgcctcc ttaaagacat ggttttgggt cctgccgtct cacccttact catgccaatg 360ccaataccag cctggagagg gaatgcccgc gatggtatcg cccacggtga ccctttttgc 420gcgagaacat gcctccgtca aggctgtacc cgatgcgaac tgggtgctca aaggggtccc 480gacattcaac tattgtgcca gatgacgaag gaccagacaa aggggaggac ggccattgga 540tggccgcatg cgaaccggtg ccgacggtat gccaggtatg ctttcggtgc cgccgcgtcg 600ttctgctaat gtttacgata ataattcacg gcggtgtata aaagccacct ctccagtgca 660acctttcttc caagacacag tcttcctctc aacagtcttc tagctgcaat ggccttcaag 720caactcctcg ctgcgctctc cgtcgcaatt ttcctaggca ccgcccaggg tatgctctct 780acgccgtacc tccgaccgcc accgtcctga ctgcttttca ggtgcgatta ccagacgtgt 840tgcctgcccc gacggggtga acactgcgac gaacgccgca tgctgcgcct tgttcgccgt 900tcgcgacgac atccaggcca acatgttcga cggcggccag tgcaacgacg ttgctcacca 960gtcgctccgt ctgtgagtac aacggccaac aggctgcatt cgccatactc acccatcgac 1020tcaggacttt ccacgatgca gtcgcgttct ctcccgcgct cactgcacaa ggccagttcg 1080ggtaagtttt cctccatacc acacaaagcg ttggctgatt agacttatca tcagaggaaa 1140cggtgctgat ggttctatca tcaccttcgg tgatatcgag acggccttcc accccaacat 1200cggcctcgac gaaatcgtcg ccatccagaa gccgttcatc gcgaagcaca acatgacagc 1260tggcgacttg tgagtctctt gcagatagac tatcatatct tcaactcagt cattacttcg 1320gcgattagcc tccacttcgc tggtgcgatt gctacgacca actgccctgg tgctcccacc 1380atcagcttcc tcttgggtaa attatatcca cattatcatc tcattattat ccaactaatt 1440attgcacctc aggccgtccc gaggctactc aggctgctcc tgatggtctc gttccagagc 1500cgttccgtgc gtgtgccttt ctttacagct gagcttcact aatgtcggta ccaaaaccag 1560acactgtcga ccagattctg gcccgcatga acgacgcact ggaatttgac gagctcgaga 1620ctgtttgggc tctcattgcg tgagtaaaat ttttatcagt acaatgctgt tgctgactga 1680cctccaaacc agccacacca ctggtgccgc caacgatatc gacacaacca tcccgcgcac 1740ccccttcgac tctacgccga cgctcttcga ctcccagttc ttcatcgaga cccagctcaa 1800gggcaccttg ttccccgggt aagcagaggc ttgtacatta caccgcgcgt agtggactga 1860cacatgcatt agcactggtg gagacaacgg cgcaaacaca ggcgaggtca tgtccggtct 1920ggccggcgag atgcgtctgc agtccgactt cctcatcgcc cgcgacgcga ggacagcctg 1980cgaatggcaa tcgttctccg gcaacatgcc caagctccag aaccgcttcc agttcgtcct 2040cgagaccttt gctgtcgtcg gccaggacca gaccaacatg atcgactgct ccgaggtcat 2100ccccgtcccc gtcgacctca ccgacgagca ggctgctggc ttcttccctc ccggaaagac 2160tctcgatgat gttgagggag ctgtgagttc tctttttctt ttctcgtgtg cctacgactg 2220attgtacatc gttcagtgcg ccgacactcc gttcccctcg ttcgctaccg cccctggccc 2280cgccactgct atccccgccg tgtaagtttc aaagcaattg tgctcttgta ttgcgagcta 2340atagccccta tagcccgtcg tccccggtca actcacctaa gtagatgtga ggttcatcgg 2400atggaatatc actcgacaac ggcatggata tactgkttaa ggatyytwag tggkgttttg 2460tattatatag tgaccgtgna tgtatgcag 2489 2 370 PRT Bjerkandera adusta 2 MetAla Phe Lys Gln Leu Leu Ala Ala Leu Ser Val Ala Ile Phe Leu 1 5 10 15Gly Thr Ala Gln Gly Met Leu Ser Thr Pro Arg Val Ala Cys Pro Asp 20 25 30Gly Val Asn Thr Ala Thr Asn Ala Ala Cys Cys Ala Leu Phe Ala Val 35 40 45Arg Asp Asp Ile Gln Ala Asn Met Phe Asp Gly Gly Gln Cys Asn Asp 50 55 60Val Ala His Gln Ser Leu Arg Leu Thr Phe His Asp Ala Val Ala Phe 65 70 7580 Ser Pro Ala Leu Thr Ala Gln Gly Gln Phe Gly Gly Asn Gly Ala Asp 85 9095 Gly Ser Ile Ile Thr Phe Gly Asp Ile Glu Thr Ala Phe His Pro Asn 100105 110 Ile Gly Leu Asp Glu Ile Val Ala Ile Gln Lys Pro Phe Ile Ala Lys115 120 125 His Asn Met Thr Ala Gly Asp Phe Leu His Phe Ala Gly Ala IleAla 130 135 140 Thr Thr Asn Cys Pro Gly Ala Pro Thr Ile Ser Phe Leu LeuGly Arg 145 150 155 160 Pro Glu Ala Thr Gln Ala Ala Pro Asp Gly Leu ValPro Glu Pro Phe 165 170 175 His Thr Val Asp Gln Ile Leu Ala Arg Met AsnAsp Ala Leu Glu Phe 180 185 190 Asp Glu Leu Glu Thr Val Trp Ala Leu IleAla His Thr Thr Gly Ala 195 200 205 Ala Asn Asp Ile Asp Thr Thr Ile ProArg Thr Pro Phe Asp Ser Thr 210 215 220 Pro Thr Leu Phe Asp Ser Gln PhePhe Ile Glu Thr Gln Leu Lys Gly 225 230 235 240 Thr Leu Phe Pro Gly ThrGly Gly Asp Asn Gly Ala Asn Thr Gly Glu 245 250 255 Val Met Ser Gly LeuAla Gly Glu Met Arg Leu Gln Ser Asp Phe Leu 260 265 270 Ile Ala Arg AspAla Arg Thr Ala Cys Glu Trp Gln Ser Phe Ser Gly 275 280 285 Asn Met ProLys Leu Gln Asn Arg Phe Gln Phe Val Leu Glu Thr Phe 290 295 300 Ala ValVal Gly Gln Asp Gln Thr Asn Met Ile Asp Cys Ser Glu Val 305 310 315 320Ile Pro Val Pro Val Asp Leu Thr Asp Glu Gln Ala Ala Gly Phe Phe 325 330335 Pro Pro Gly Lys Thr Leu Asp Asp Val Glu Gly Ala Cys Ala Asp Thr 340345 350 Pro Phe Pro Ser Phe Ala Thr Ala Pro Gly Pro Ala Thr Ala Ile Pro355 360 365 Ala Val 370 3 3653 DNA Bjerkandera adusta 3 cagcggaataaccttagtca tactgagtac acggacgtgt gtatgtgcct gtagaggctt 60 ctcggcggccatactttgag ttccgcccac acggagtgac agaaggagac ctggtcctgg 120 accaaagcaagaccgctgtt gtctggatcg gaattcgaca ggactcaatt ttgaaacaga 180 agtttcggagcatagttggt gaaagtatga gtctcgtata ttcctggatg gaattacagg 240 ccctttctcgcggtaatgct tgcttactct tatgagaata aatggtggcg ttcggaaaat 300 gccgctacctgttacttacc gtgggatttt gttgcacctt actatcacac gtcaaaagtg 360 gactcttgcgtctttccgtc tctctcccaa ccaaatcgac tcggactaga gaccaacggg 420 cgtacgacaacatacatcga tcttcacatg gacaatgttg cagactgttc aaggtcactc 480 cgtcccaactcccgggtagg ctcgaaccgg tgatacttac ctcgatcgtg cgagagaaca 540 agtacttataatagacttag ggtcgtcgcc ggcaggtacg atgatggtcg gcatctcagt 600 ctctcgagggtgctatagtt atgtctgacg gtacctggcc ctgacatggc acttcgcgcg 660 aaacttgcgtcgtgaggtca tggccgcctc ggctcgtttg acaactgtgt acgcaaaggt 720 gagtactcggggagccgtct ggcttcacca gctactcctg ccgcattact aagtctccga 780 agcgcgccgatatgtagctt gtgttttgga tgagaacgct cgggcaaaac ggcgactagc 840 gacatcgtatcgacgaccgg cggccgcggt ctctaaaggc catatggaca taatcctcga 900 atgatcgaaaagacgggagt tatttttgtt ttctgtgcgt catacgacgg tttacttgtt 960 gtccgagacctcttctcacg acgcgttgcc tagtcgagaa tgaacgttct cactttcctt 1020 ttgcaggatttggctcagat cagtgatggc cgcggcagcc caactcggag aagtaggaat 1080 ttgcacttctgtgttcaaag aacactgctg caagactact tacccagccg ccacgaagcc 1140 gttgaaaagcctaaccatag tctgttttcc atgtctaggc atcgcaagag gcggcagaac 1200 gagacctcgtcttcggttct gacatgacgg accgacaaac gagtgtacct caaacatggt 1260 cctggcgccattcatagcgg taagatattt gattctcgcg ttaaacctgt accaatgttt 1320 tggttcattcccagaaaccg tgcgatgcgg gcagtacagg tcccagcctt cgcgtgaagc 1380 gagtactacgcagcaccacc gactgcggct catcatacgt ctatgtggcc tgccgactac 1440 tatcaacgaccgcggatgcg tgcccacgca acggcaccga cggcgctgct gtccagctcc 1500 atcaacggtcccgcgcgaag caggactatg agctttttgt gcagaagaga cacagccctc 1560 tgttgattttgccgtaaggc agcagtggac ggctcgcgcg tggggagtcg ccgaggttat 1620 tttcggctcgtggtacggga aagttctgtc tacggctgtc gagacatgga aatccgtacc 1680 actggactgcgaggctggag gcgaaccgtg gaagagggcc ggagtgccct caaacgagga 1740 tattctcattggccgcagca aagggaacat cttgagagac aatgtggcgc tgcaagctag 1800 aggcatacttctgcgaagta taaaagctgc tagagtagtt gggaccatcc tcaggacatc 1860 cgtcttctaccctctactca gtcaaaccag caatggcctt caagcagctc ctcgccactg 1920 tctctctcgccttctccctc accgctgtcg aaggtttgtg gcgaattact ctgccagcca 1980 cttgtgctcatctatcgcgc tttagccgcc cttacccgcc gggttgcttg ccccgatggc 2040 gtgaacaccgcgacgaacgc ggcctgctgc tctctgttcg ccatccgtga cgatcttcaa 2100 caaagcctgttcgacaacgg cggatgtggc gaagatgttc acgagtctct ccgtctgtga 2160 gtatacgaccagccccgaat cccgacccaa aatctaaccg gatatactag caccttccac 2220 gacgctatcggtatttctcc cgccatcgcg gcaaccggaa agttcgggtg cgtatacatc 2280 caaaatatgatgtctcctcg cgttctgact agtcgcgcag aggtggaggt gccgacggct 2340 ctattgccatcttcgaggac atcgagacca acttccacgc gaacttgggc gtcgacgaga 2400 tcatcaacgagcagaggccc atcctggcca gacacaacat caccaccgct gacttgttcg 2460 tcgcttcctgatcattctcc actatactgc taaccgatcg tttagcattc agtttgctgg 2520 tgcagtcggcgtgagcaact gccccggtgc ccctcagctc gagttcctct tcggtaagcg 2580 aaaccgtctttcatcataac acatctactc acgcgactgt acaggccgca cggacgccac 2640 ccagcccgcccccgacctca ccgtccccga gccttccgat accgtcgact ccatcatcgc 2700 tcgcttcgctgacgctggag gcttcacccc cgcggagatc gttgcccttc tcgcctcgta 2760 aggttatttcatactgcaaa aagcatcccg ctgatacacg ccacctatgc agccacaccg 2820 ttgccgcggccgaccacgtc gaccccacca ttccgggaac tccattcgac tcgaccgcct 2880 ctaccttcgactcccagttc ttcgtcgaga cgctgctcaa gggcacgctc ttcccggtac 2940 gcctaccttcgatccgactt ctcccttgca tttctgacat tagcacaaca gaacttcggg 3000 caacgtcggagaggtgatgt cccccatcgc gggtgagatg cgtctgcagt ccgacttcga 3060 gctcgcacaagactctcgta ctgcttgcga gtggcagtcg ttcgtcagtg cgtgccctct 3120 ccttccctttcgccccgccc ggattctctg accgtacacc agacaaccag gacaagatca 3180 agaccgcgtttgctgccgcg ttcgccaaga tggccaccct cggaaatgac aggagccaga 3240 tggtcgactgctccgaggtg ctgcccaggg tctcgaccgc cactctcccg cccgcgcacc 3300 tccccgccggcaagacgctc gccgacgtcc agcaggctgt acgcacttca tattcactct 3360 gtgcgcgagaagttgagctg acgatacctg cttcagtgcg ccgacacccc cttcccgtct 3420 ctctctgccgaccccggccc ggccaccact gtcccccctg tgtaagtgtt atacgataca 3480 attccctcagcgacggtgtg ctaacgtgat aaattcgtgc agcccgcctt cctaagttgc 3540 catctagtcagtcgagacgg tatatcgact gaggcgtcgt ctcatctgtc ggaagtagaa 3600 gttctgcgaatgtatctatc tgttgattcg aatggggatc cgcttttgtg aac 3653 4 366 PRTBjerkandera adusta 4 Met Ala Phe Lys Gln Leu Leu Ala Thr Val Ser Leu AlaPhe Ser Leu 1 5 10 15 Thr Ala Val Glu Ala Ala Leu Thr Arg Arg Val AlaCys Pro Asp Gly 20 25 30 Val Asn Thr Ala Thr Asn Ala Ala Cys Cys Ser LeuPhe Ala Ile Arg 35 40 45 Asp Asp Leu Gln Gln Ser Leu Phe Asp Asn Gly GlyCys Gly Glu Asp 50 55 60 Val His Glu Ser Leu Arg Leu Thr Phe His Asp AlaIle Gly Ile Ser 65 70 75 80 Pro Ala Ile Ala Ala Thr Gly Lys Phe Gly GlyGly Gly Ala Asp Gly 85 90 95 Ser Ile Ala Ile Phe Glu Asp Ile Glu Thr AsnPhe His Ala Asn Leu 100 105 110 Gly Val Asp Glu Ile Ile Asn Glu Gln ArgPro Ile Leu Ala Arg His 115 120 125 Asn Ile Thr Thr Ala Asp Phe Ile GlnPhe Ala Gly Ala Val Gly Val 130 135 140 Ser Asn Cys Pro Gly Ala Pro GlnLeu Glu Phe Leu Phe Gly Gly Arg 145 150 155 160 Thr Asp Ala Thr Gln ProAla Pro Asp Leu Thr Val Pro Glu Pro Ser 165 170 175 Asp Thr Val Asp SerIle Ile Ala Arg Phe Ala Asp Ala Gly Gly Phe 180 185 190 Thr Pro Ala GluIle Val Ala Leu Leu Ala Ser His Thr Val Ala Ala 195 200 205 Ala Asp HisVal Asp Pro Thr Ile Pro Gly Thr Pro Phe Asp Ser Thr 210 215 220 Ala SerThr Phe Asp Ser Gln Phe Phe Val Glu Thr Leu Leu Lys Gly 225 230 235 240Thr Leu Phe Pro His Asn Arg Thr Ser Gly Asn Val Gly Glu Val Met 245 250255 Ser Pro Ile Ala Gly Glu Met Arg Leu Gln Ser Asp Phe Glu Leu Ala 260265 270 Gln Asp Ser Arg Thr Ala Cys Glu Trp Gln Ser Phe Val Asn Asn Gln275 280 285 Asp Lys Ile Lys Thr Ala Phe Ala Ala Ala Phe Ala Lys Met AlaThr 290 295 300 Leu Gly Asn Asp Arg Ser Gln Met Val Asp Cys Ser Glu ValLeu Pro 305 310 315 320 Arg Val Ser Thr Ala Thr Leu Pro Pro Ala His LeuPro Ala Gly Lys 325 330 335 Thr Leu Ala Asp Val Gln Gln Ala Cys Ala AspThr Pro Phe Pro Ser 340 345 350 Leu Ser Ala Asp Pro Gly Pro Ala Thr ThrVal Pro Pro Val 355 360 365 5 4810 DNA Bjerkandera adusta 5 cgcatcttcatccccggcac gaagggcctg cagcttgccc gcgtaaattt cgcgcgccat 60 acggtccaaagtatctataa catccttcaa ttcgtgtatg gcccggcttg gcgcgtgctc 120 cacgaggaatcgctttaccc gcgatgatgg gatatacttc acgagtggag aaaagaaagc 180 aaagacagcgcattttgcga gggccggcct ggaaagcgct agatgtgagt tccgagatcc 240 actctttctctggccgatgc tcacactatg tgcttgactg cttccgcata ctcgttgcga 300 gtgtcttcgacaaggctgtc aaaggagtag cccataccgc cttgtccaat aagctccaat 360 gcagcacggcccaatccatc taagcatgtc caattcgaca ctttctgatc ccttgacctg 420 ccgcgcgatgccatccttga gctgagtcaa gttgattaat tatatgccgg tacggggtac 480 cggcaagacacctacgcggt ggacggtctc gtagaacaca ggcgttaggt tcttcatatt 540 tcccagcgagaaaacaggat tgagcagctt cctttggcgg cggtggtgat gacctagaac 600 ggtccgacgcatggtcaagg tgaacggccg atgcagactc gtaggacact gcttaccatg 660 cgtggataacagcccagggc cgaagacaat gtttcgatat ctgaaggatg gtgcacacgg 720 ttcagtgcacggtcggagat ggagaagcgg aattggataa ctcacgcgag tacgtagttt 780 ggttccggatacatgtcctg atccttgaga acgatataat gcagcgcctt cgggtcaaac 840 acgtaaagcccccttcgctg aaaaaacaaa gggattcagc ggaaagtcgc acggtagaga 900 ggatcacgcacgccgaacaa cgaacggaac ttgaccactg caccatagtt gtcgctgaga 960 tggtctatgaacgccatacc gccttctcgc tgaaagagct gtttgaagtt tcctgagagg 1020 agtcaaagtctagcggcgcc gagattgtac gactctcgac cttacctgtg aggaatgaag 1080 gggacggaggcccaggtatc ttgtctagag gcgattccac ggtcaacgca cggaagacct 1140 tccatatcagccatgccaca acaaaggaaa tgcccgccga tggtgcggag agcgtcatgg 1200 tcgtagtagaaagtgcgcag acgtggggat ggcgagtctg tttcgccttg atgtccgacc 1260 gtagccgtcgggtgagcgtt agcgaagcag cgtcatcact gtcgcgtatt ccttgttagt 1320 aacacaagatctccgcggtt tcaatgtggc tcttgctgta ggggcccagg gagggtgggg 1380 ggtgttcaacgaccgcagta gcttcggaaa cccctcctcc cacaccaagt gagcttctga 1440 catgaattaatcgtcaagca ctacccgcag ctgtttgaag tacaaaggta ggtgataatc 1500 aggaaacctggagtgtgaga gaccccatta aagtgactga ccgacggccg cgcgatatcg 1560 tcttcaggctgttgtttgat tgttaaaaag actatttcct gcagcgaaac tttatagaac 1620 ccatcgttgctgcggtcatg atcgaagtta acaggtgcct ggtcaaaggt caggcccatt 1680 tgtagcatttgttggacata gtgggcacca accttggcgt tgaagacaag agaacgaatg 1740 acgattcgggaattggatca gcatgctgga cgtctccgcc agtattatct acactccgca 1800 gggaagccgccggggccgaa tgttgcggag ccgagcggac taggaacagg cgcgcggaga 1860 ccgccggtacactcgtattc ttcctccgca gcgagttgtt tgaacaggac tgtcgcttct 1920 gatttgtttctgtaccagat acttctatcc gattgcgcgg cgtcggatgt gctcgctgac 1980 cttcatccagccctctgtgc cctggtttta caggttattt ttgaagcccg aacttccatc 2040 tcctgcttctacggactgcc ccgtacaaat gtgaagagcg acatcgcgct ccgcaggcct 2100 ctgtgacctccaggaattct ctacagcggg accctgcagg tacgtgtgcg acaatcactg 2160 cggcaccaagtcggcttgcc aacgattgcc tttttctata gaccgagtgt cgggtcggtc 2220 ccgcgctggcgcacccagac ctcctccgga atgtagttcg tgctgtgcaa acatgcacca 2280 ctggaaactccgcctcgcgt taggactagc gccaaatgcg gtcgcctctc gcgagtacct 2340 attgcactgcttgcacaggg cttctctcgc gtatcaagcc actggcgaat acatcacaag 2400 cgggcccgtgagctgcgttg cagcaaatgc gcagtctgta gagtccagct ctcgaataca 2460 aacagactgcgcgtggctcg ggcaggccgg tcgcatcatg aaacgagggt cagatgccgg 2520 cagcgacgaccacggtccgc ggacgaggtt ggggggatga tgcccgactg gagaatggcg 2580 gtgctcctattgggcccggg cgctgttggc ttggctggtc ggctccgggg tggtcgatct 2640 ccaccacgtttatcgaagcc cgtcgcgctg gcggatcaca gatccaccac cataccactt 2700 ccagtataaagagcgccggg tatgcaagcg aacacctcat cgtcccttcc cttctctcct 2760 ttcctctaatcccctccttc gttggtcgtc gacatggtgt tctacagact ctcctccctc 2820 cttgtgtctgttgctgctat ccacgccgca gccggtacgc cacataatgg ctcccccctc 2880 ccgatccacgctgaccagct tgctaggtgc tctgacgcgc cgtgtcgcat gcccggatgg 2940 cgtgaacaccgcgaccaacg cggcgtgctg ccccttgtac gccgtccgcg atgacatgca 3000 ggccaacctgtatgatggtg gcgcgtgcaa cgccgaggtg catgagtccc tccgcctgtg 3060 agtacccagcttgctttcgg tgttgcacaa agctcatcta gtgccagcac attccacgac 3120 gccattggtacgtcttgctt agatttcttc caacggtgtc ttacgatatt ctacaggcta 3180 ctccccagccctcgccgccg ccggctcatt cgcaggtgga ggagctgacg gctctatcct 3240 taccttcagcgatgttgaag cggccttctt tgccaacgcg ggtctcgacg agatgatcga 3300 gctccagaagccatacatca ccaagtacaa catgactcct ggcgatgtgt acgtaccagg 3360 aacctctttggcgatattat actgaagcgt ctctatagcg ttcaatttgc tggcgccgtc 3420 ggtctcagtaactgcccagg tgctccgcaa ctggagttcc ttctcggtac gtcacgcaat 3480 aaatcagaccgcgaaagcca cgttctgatc atcgcccagg tcgtactgcc gctacggccg 3540 cgtcgcccacaggcctcatt cccgcaccct ttgacacggt cgatgcgatc attgcgcgct 3600 tcgccgacgtcgacttcagc gttgacgaga ttgtagcgct gttggcatcg taagcacgcc 3660 ccctttgcttggcgagcatc atgattaacg acgtctcact tgcaggcact cggtcgccgc 3720 tgcaagccacatcgacacca ccgttcctga gtcgccgctc gactcgaccc ctggcgtctt 3780 cgacacccagttcttcgtcg aaacctcgct caacggcacc atgtaccctg gtacctctgg 3840 aaacatcggcgaggccctgt cagcgattgc gggagagctt cgcctgctct cggaccatga 3900 gctcgcgcgtgactcgcgca ctgcctgcga gtggcagtcc tacgtcagta cgtaccctgt 3960 ccttcggttccgttgacggt ttccatttat gctttacgtg tgcagacaac cagtccaaga 4020 tccagagcgcgttccgtgct gccatggcga ggatggccgt catcggccag gactcctcga 4080 ccatgattgactgcaccgaa gtcatcccca ccgcatcgtc cttcacctcc gccgcgttta 4140 tccccgccggtctcacctac gctgacatcg aacagtcgtg cgactccact cccttcccca 4200 ccctttctgtcgttgccggc gcggccacgt ccgtcgctgc cgttgcgtga gttgtttcgg 4260 ttcctctcaaattccacata gtactgacaa aatatttaga aaatcataaa ctgcccacac 4320 cggcaacccctggcttctat tctatctttt tgggttaata tggacttctt gaacacttgt 4380 ggttgaaattggattgaatt agtactgtcg ctacctgccc ggacctttgt aaacactgtc 4440 tgtctctacgagtaaatagc ccgctccaaa accgtctatc tatagaggta tccactgcca 4500 aatgtcatcgctattatctg tctacatttt gctgcgcgac ataaaaaccc gatatggact 4560 tctcgtcgacattgtggcgc cgcagcaaag cggtctaacc gcaaatggca ggtatgtttt 4620 gaaatctcggcctgctgttg tacctcaaaa tactcaagta cgttctcaca tgttcgcgcg 4680 atgaccgtacagcaactcca cggttgtggc gaagcagtcg aaaattaaat ggagcaggca 4740 ccactcacttggcatctgcc atgtcacggt cctctggagt ctgagccaag acaaaactgg 4800 atcggggcat4810 6 362 PRT Bjerkandera adusta 6 Met Val Phe Tyr Arg Leu Ser Ser LeuLeu Val Ser Val Ala Ala Ile 1 5 10 15 His Ala Ala Ala Gly Ala Leu ThrArg Arg Val Ala Cys Pro Asp Gly 20 25 30 Val Asn Thr Ala Thr Asn Ala AlaCys Cys Pro Leu Tyr Ala Val Arg 35 40 45 Asp Asp Met Gln Ala Asn Leu TyrAsp Gly Gly Ala Cys Asn Ala Glu 50 55 60 Val His Glu Ser Leu Arg Leu ThrPhe His Asp Ala Ile Gly Tyr Ser 65 70 75 80 Pro Ala Leu Ala Ala Ala GlySer Phe Ala Gly Gly Gly Ala Asp Gly 85 90 95 Ser Ile Leu Thr Phe Ser AspVal Glu Ala Ala Phe Phe Ala Asn Ala 100 105 110 Gly Leu Asp Glu Met IleGlu Leu Gln Lys Pro Tyr Ile Thr Lys Tyr 115 120 125 Asn Met Thr Pro GlyAsp Val Val Gln Phe Ala Gly Ala Val Gly Leu 130 135 140 Ser Asn Cys ProGly Ala Pro Gln Leu Glu Phe Leu Leu Gly Arg Thr 145 150 155 160 Ala AlaThr Ala Ala Ser Pro Thr Gly Leu Ile Pro Ala Pro Phe Asp 165 170 175 ThrVal Asp Ala Ile Ile Ala Arg Phe Ala Asp Val Asp Phe Ser Val 180 185 190Asp Glu Ile Val Ala Leu Leu Ala Ser His Ser Val Ala Ala Ala Ser 195 200205 His Ile Asp Thr Thr Val Pro Glu Ser Pro Leu Asp Ser Thr Pro Gly 210215 220 Val Phe Asp Thr Gln Phe Phe Val Glu Thr Ser Leu Asn Gly Thr Met225 230 235 240 Tyr Pro Gly Thr Ser Gly Asn Ile Gly Glu Ala Leu Ser AlaIle Ala 245 250 255 Gly Glu Leu Arg Leu Leu Ser Asp His Glu Leu Ala ArgAsp Ser Arg 260 265 270 Thr Ala Cys Glu Trp Gln Ser Tyr Val Asn Asn GlnSer Lys Ile Gln 275 280 285 Ser Ala Phe Arg Ala Ala Met Ala Arg Met AlaVal Ile Gly Gln Asp 290 295 300 Ser Ser Thr Met Ile Asp Cys Thr Glu ValIle Pro Thr Ala Ser Ser 305 310 315 320 Phe Thr Ser Ala Ala Phe Ile ProAla Gly Leu Thr Tyr Ala Asp Ile 325 330 335 Glu Gln Ser Cys Asp Ser ThrPro Phe Pro Thr Leu Ser Val Val Ala 340 345 350 Gly Ala Ala Thr Ser ValAla Ala Val Ala 355 360 7 26 DNA Artificial Sequence Bjerkandera adusta7 gtngcntgyc cngayggngt naayac 26 8 20 DNA Artificial SequenceBjerkandera adusta 8 tgnggngcnc cnggrcartt 20 9 26 DNA ArtificialSequence Bjerkandera adusta 9 gtngcntgyc cngayggngt naayac 26 10 19 DNAArtificial Sequence Bjerkandera adusta 10 tgccaytcrc angcngtnc 19

What is claimed is:
 1. An isolated polypeptide having peroxidaseactivity, selected from the group consisting of: (a) a polypeptidehaving an amino acid sequence which has at least 75% identity with aminoacids 22 to 370 of SEQ ID NO: 2, amino acids 22 to 366 of SEQ ID NO: 4,or amino acids 19 to 362 of SEQ ID NO: 6; (b) a polypeptide which isencoded by a nucleic acid sequence which hybridizes under medium-highstringency conditions with (i) nucleotides 772 to 2302 of SEQ ID NO: 1,nucleotides 2008 to 3462 of SEQ ID NO: 3, or nucleotides 2848 to 4247 ofSEQ ID NO: 5, (ii) the cDNA sequence contained in nucleotides 772 to2302 of SEQ ID NO: 1, nucleotides 2008 to 3462 of SEQ ID NO: 3, ornucleotides 2848 to 4247 of SEQ ID NO: 5, (iii) a subsequence of (i) or(ii) of at least 100 nucleotides, or (iv) a complementary strand of (i),(ii), or (iii); (c) a variant of the polypeptide having an amino acidsequence of SEQ ID NO: 2 comprising a substitution, deletion, and/orinsertion of one or more amino acids; (d) an allelic variant of (a) or(b); and (e) a fragment of (a), (b), or (d) that has peroxidaseactivity.
 2. The polypeptide of claim 1, having an amino acid sequencewhich has at least 75% identity with amino acids 22 to 370 of SEQ ID NO:2, amino acids 22 to 366 of SEQ ID NO: 4, or amino acids 19 to 362 ofSEQ ID NO:
 6. 3. The polypeptide of claim 2, having an amino acidsequence which has at least 80% identity with amino acids 22 to 370 ofSEQ ID NO: 2, amino acids 22 to 366 of SEQ ID NO: 4, or amino acids 19to 362 of SEQ ID NO:
 6. 4. The polypeptide of claim 3, having an aminoacid sequence which has at least 85% identity with amino acids 22 to 370of SEQ ID NO: 2, amino acids 22 to 366 of SEQ ID NO: 4, or amino acids19 to 362 of SEQ ID NO:
 6. 5. The polypeptide of claim 4, having anamino acid sequence which has at least 90% identity with amino acids 22to 370 of SEQ ID NO: 2, amino acids 22 to 366 of SEQ ID NO: 4, or aminoacids 19 to 362 of SEQ ID NO:
 6. 6. The polypeptide of claim 5, havingan amino acid sequence which has at least 95% identity with amino acids22 to 370 of SEQ ID NO: 2, amino acids 22 to 366 of SEQ ID NO: 4, oramino acids 19 to 362 of SEQ ID NO:
 6. 7. The polypeptide of any ofclaims 1-6, comprising the amino acid sequence of SEQ ID NO: 2, SEQ IDNO: 4, or SEQ ID NO:
 6. 8. The polypeptide of any of claims 1-7,consisting of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, orSEQ ID NO: 6, or a fragment thereof having peroxidase activity.
 9. Thepolypeptide of claim 8, consisting of the amino acid sequence of SEQ IDNO: 2, SEQ ID NO: 4, or SEQ ID NO:
 6. 10. The polypeptide of claim 9,which consists of amino acids 22 to 370 of SEQ ID NO: 2, amino acids 22to 366 of SEQ ID NO: 4, or amino acids 19 to 362 of SEQ ID NO:
 6. 11.The polypeptide of claim 1, which is encoded by a nucleic acid sequencewhich hybridizes under medium-high stringency conditions with (i)nucleotides 772 to 2302 of SEQ ID NO: 1, nucleotides 2008 to 3462 of SEQID NO: 3, or nucleotides 2848 to 4247 of SEQ ID NO: 5, (ii) the cDNAsequence contained in nucleotides 772 to 2302 of SEQ ID NO: 1,nucleotides 2008 to 3462 of SEQ ID NO: 3, or nucleotides 2848 to 4247 ofSEQ ID NO: 5, (iii) a subsequence of (i) or (ii) of at least 100nucleotides, or (iv) a complementary strand of (i), (ii), or (iii). 12.The polypeptide of claim 11, which is encoded by a nucleic acid sequencewhich hybridizes under medium-high stringency conditions with (i)nucleotides 772 to 2302 of SEQ ID NO: 1, nucleotides 2008 to 3462 of SEQID NO: 3, or nucleotides 2848 to 4247 of SEQ ID NO: 5, (ii) the cDNAsequence contained in nucleotides 772 to 2302 of SEQ ID NO: 1,nucleotides 2008 to 3462 of SEQ ID NO: 3, or nucleotides 2848 to 4247 ofSEQ ID NO: 5, or (iii) a complementary strand of (i) or (ii).
 13. Thepolypeptide of claim 1, which is encoded by a nucleic acid sequencewhich hybridizes under high stringency conditions with (i) nucleotides772 to 2302 of SEQ ID NO: 1, nucleotides 2008 to 3462 of SEQ ID NO: 3,or nucleotides 2848 to 4247 of SEQ ID NO: 5, (ii) the cDNA sequencecontained in nucleotides 772 to 2302 of SEQ ID NO: 1, nucleotides 2008to 3462 of SEQ ID NO: 3, or nucleotides 2848 to 4247 of SEQ ID NO: 5,(iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv)a complementary strand of (i), (ii), or (iii).
 14. The polypeptide ofclaim 13, which is encoded by a nucleic acid sequence which hybridizesunder high stringency conditions with (i) nucleotides 772 to 2302 of SEQID NO: 1, nucleotides 2008 to 3462 of SEQ ID NO: 3, or nucleotides 2848to 4247 of SEQ ID NO: 5, (ii) the cDNA sequence contained in nucleotides772 to 2302 of SEQ ID NO: 1, nucleotides 2008 to 3462 of SEQ ID NO: 3,or nucleotides 2848 to 4247 of SEQ ID NO: 5, or (iii) a complementarystrand of (i) or (ii).
 15. The polypeptide of claim 1, wherein thepolypeptide is a variant of the polypeptide having an amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 comprising asubstitution, deletion, and/or insertion of one or more amino acids. 16.The polypeptide of claim 1, which is encoded by the nucleic acidsequence contained in plasmid pBM37-7 which is contained in E. coli NRRLB-30280, plasmid pBM38-1 which is contained in E. coli NRRL B-30281, orplasmid pBM39-1 which is contained in E. coli NRRL B-30282.
 17. Thepolypeptide of any of claims 1-16 which has at least 20% of theperoxidase activity of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO:
 6. 18.An isolated nucleic acid sequence comprising a nucleic acid sequencewhich encodes the polypeptide of any of claims 1-17.
 19. An isolatednucleic acid sequence comprising a nucleic acid sequence having at leastone mutation in the mature polypeptide coding sequence of SEQ ID NO: 1,SEQ ID NO: 3, or SEQ ID NO: 5, in which the mutant nucleic acid sequenceencodes a polypeptide consisting of amino acids 22 to 370 of SEQ ID NO:2, amino acids 22 to 366 of SEQ ID NO: 4, or amino acids 19 to 362 ofSEQ ID NO: 6, respectively.
 20. An isolated nucleic acid sequenceproduced by (a) hybridizing a DNA under medium-high stringencyconditions with (i) nucleotides 772 to 2302 of SEQ ID NO: 1, nucleotides2008 to 3462 of SEQ ID NO: 3, or nucleotides 2848 to 4247 of SEQ ID NO:5, (ii) the cDNA sequence contained in nucleotides 772 to 2302 of SEQ IDNO: 1, nucleotides 2008 to 3462 of SEQ ID NO: 3, or nucleotides 2848 to4247 of SEQ ID NO: 5, (iii) a subsequence of (i) or (ii) of at least 100nucleotides, or (iv) a complementary strand of (i), (ii), or (iii); and(b) isolating the nucleic acid sequence.
 21. The isolated nucleic acidsequence of claim 20 produced by (a) hybridizing a DNA under highstringency conditions with (i) nucleotides 772 to 2302 of SEQ ID NO: 1,nucleotides 2008 to 3462 of SEQ ID NO: 3, or nucleotides 2848 to 4247 ofSEQ ID NO: 5, (ii) the cDNA sequence contained in nucleotides 772 to2302 of SEQ ID NO: 1, nucleotides 2008 to 3462 of SEQ ID NO: 3, ornucleotides 2848 to 4247 of SEQ ID NO: 5, (iii) a subsequence of (i) or(ii) of at least 100 nucleotides, or (iv) a complementary strand of (i),(ii), or (iii); and (b) isolating the nucleic acid sequence.
 22. Anucleic acid construct comprising the nucleic acid sequence of claim 18operably linked to one or more control sequences that direct theproduction of the polypeptide in a suitable expression host.
 23. Arecombinant expression vector comprising the nucleic acid construct ofclaim
 22. 24. A recombinant host cell comprising the nucleic acidconstruct of claim
 22. 25. A method for producing a mutant nucleic acidsequence, comprising (a) introducing at least one mutation into themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQID NO: 5, wherein the mutant nucleic acid sequence encodes a polypeptideconsisting of amino acids 22 to 370 of SEQ ID NO: 2, amino acids 22 to366 of SEQ ID NO: 4, or amino acids 19 to 362 of SEQ ID NO: 6,respectively; and (b) recovering the mutant nucleic acid sequence.
 26. Amutant nucleic acid sequence produced by the method of claim
 25. 27. Amethod for producing a polypeptide, comprising (a) cultivating a straincomprising the mutant nucleic acid sequence of claim 26 encoding thepolypeptide to produce a supernatant comprising the polypeptide; and (b)recovering the polypeptide.
 28. A method for producing the polypeptideof any of claims 1-17 comprising (a) cultivating a strain to produce asupernatant comprising the polypeptide; and (b) recovering thepolypeptide.
 29. A method for producing the polypeptide of any of claims1-17 comprising (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleic acid sequence encoding the polypeptideunder conditions suitable for production of the polypeptide; and (b)recovering the polypeptide.
 30. A method for producing a polypeptidecomprising (a) cultivating a host cell under conditions conducive forproduction of the polypeptide, wherein the host cell comprises a mutantnucleic acid sequence having at least one mutation in the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO:5, wherein the mutant nucleic acid sequence encodes a polypeptideconsisting of amino acids 22 to 370 of SEQ ID NO: 2, amino acids 22 to366 of SEQ ID NO: 4, or amino acids 19 to 362 of SEQ ID NO: 6,respectively, and (b) recovering the polypeptide.
 31. A method forproducing the polypeptide of any of claims 1-17 comprising (a)cultivating a homologously recombinant cell, having incorporated thereina new transcription unit comprising a regulatory sequence, an exon,and/or a splice donor site operably linked to a second exon of anendogenous nucleic acid sequence encoding the polypeptide, underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.
 32. A method for producing a mutant of acell, which comprises disrupting or deleting a nucleic acid sequenceencoding the polypeptide of any of claims 1-17 or a control sequencethereof, which results in the mutant producing less of the polypeptidethan the cell.
 33. A mutant produced by the method of claim
 32. 34. Themutant of claim 33, which further comprises a nucleic acid sequenceencoding a heterologous protein.
 35. A method for producing aheterologous polypeptide comprising (a) cultivating the mutant of claim34 under conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.
 36. A nucleic acid construct comprising agene encoding a protein operably linked to a nucleic acid sequenceencoding a signal peptide consisting of nucleotides 709 to 771 of SEQ IDNO: 1, nucleotides 1893 to 2007 of SEQ ID NO: 3, or nucleotides 2794 to2847 of SEQ ID NO: 5, wherein the gene is foreign to the nucleic acidsequence.
 37. A recombinant expression vector comprising the nucleicacid construct of claim
 36. 38. A recombinant host cell comprising thenucleic acid construct of claim
 36. 39. A method for producing a proteincomprising (a) cultivating the recombinant host cell of claim 38 underconditions suitable for production of the protein; and (b) recoveringthe protein.
 40. A method for polymerizing a lignin or lignosulfatesubstrate in solution which comprises contacting the substrate with thepolypeptide of any of claims 1-17.
 41. A method for in situdepolymerization in Kraft pulp which comprises contacting the pulp withthe polypeptide of any of claims 1-17.
 42. A method for oxidizing dyesor dye precursors which comprises contacting the dye or dye precursorwith the polypeptide of any of claims 1-17.
 43. A method of polymerizingor oxidizing a phenolic compound which comprises contacting the phenoliccompound with the polypeptide of any of claims 1-17.